Transgenic ungulates capable of human antibody production

ABSTRACT

The invention features novel methods for the production of large quantities of xenogenous antibodies, such as human antibodies. Preferably, this result is effected by inactivation of IgM heavy chain expression and, optionally, by inactivation of Ig light chain expression, and by the further introduction of an artificial chromosome which results in the expression of xenogenous antibodies (e.g., non-bovine antibodies), preferably human antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/441,503, filed May 19,2003 which claims the benefit of U.S. provisional application60/381,531, filed May 17, 2002, and U.S. provisional application60/425,056 filed Nov. 8, 2002, which are each hereby incorporated byreference. U.S. application Ser. No. 10/441,503 is also acontinuation-in part of U.S. utility application Ser. No. 09/988,115,filed Nov. 16, 2001, which claims the benefit of U.S. provisional patentapplication 60/311,625, filed Aug. 9, 2001 and U.S. provisional patentapplication 60/256,458, filed Dec. 20, 2000, and is acontinuation-in-part of U.S. utility application Ser. No. 09/714,185,filed Nov. 17, 2000, which claims the benefit of U.S. provisional patentapplication 60/166,410, filed Nov. 19, 1999. Additionally, thisapplication is a continuation-in part of U.S. utility application Ser.No. 10/032,191, filed Dec. 21, 2001, which claims the benefit of60/258,151, filed Dec. 22, 2000.

BACKGROUND OF THE INVENTION

In general, the invention provides a genetically modified ungulate thatcontains either part or all of a xenogenous antibody gene locus, whichundergoes rearrangement and expresses a diverse population of antibodymolecules. In particular, the xenogenous antibody gene may be of humanorigin. In addition, the present invention provides for an ungulate inwhich expression of the endogenous antibody genes is either reduced oreliminated. The genetic modifications in the ungulate (for example,bovine) are made using a combination of nuclear transfer and moleculartechniques. These cloned, transgenic ungulate (e.g., bovines) provide areplenishable, theoretically infinite supply of xenogenous polyclonalantibodies, particularly human antibodies, which have use, e.g., astherapeutics, diagnostics and for purification purposes. The inventionalso features methods for reducing the amount of endogenous antibody innon-human mammals, such as ungulates, that express both endogenous andxenogenous antibody. These methods increase the percentage of xenogenousB-cells and xenogenous antibody expressed by the mammals.

In 1890, Shibasaburo Kitazato and Emil Behring reported an experimentwith extraordinary results; particularly, they demonstrated thatimmunity can be transferred from one animal to another by taking serumfrom an immune animal and injecting it into a non-immune one. Thislandmark experiment laid the foundation for the introduction of passiveimmunization into clinical practice. Today, the preparation and use ofhuman immunoglobulin (Ig) for passive immunization is standard medicalpractice. In the United States alone, there is a $1,400,000,000 perannum market for human Ig, and each year more than 16 metric tons ofhuman antibody is used for intravenous antibody therapy. Comparablelevels of consumption exist in the economies of most highlyindustrialized countries, and the demand can be expected to grow rapidlyin developing countries. Currently, human antibody for passiveimmunization is obtained from the pooled serum of human donors. Thismeans that there is an inherent limitation in the amount of humanantibody available for therapeutic and prophylactic usage. Already, thedemand exceeds the supply and severe shortfalls in availability havebeen routine. Thus improved methods are needed to generate humanantibody that is free of non-human antibody for clinical applications.

For example, improved methods and enhanced transgenic animals thatproduce polyclonal antibodies of a desired species (e.g., human Igs) inthe bloodstream and which produce an array of different antibodies whichare specific to a desired antigen would be highly desirable. Mostespecially, the production of human Igs in ungulates, such as cows,would be particularly beneficial given that (1) cows could produce largequantities of antibody, (2) cows could be immunized with human or otherpathogens and (3) cows could be used to make human antibodies againsthuman antigens. The availability of large quantities of polyclonalantibodies would be advantageous for treatment and prophylaxis forinfectious disease, modulation of the immune system, removal ofundesired human cells such as cancer cells, and modulation of specifichuman molecules.

SUMMARY OF THE INVENTION

Transgenic ungulates expressing a xenogenous antibody and/or expressingdecreased levels of functional, endogenous antibody In a first aspect,the invention provides a transgenic ungulate (e.g., a bovine) having oneor more nucleic acids encoding all or part of a xenogenousimmunoglobulin (Ig) gene which undergoes rearrangement and expressesmore than one xenogenous Ig protein. In a preferred embodiment, thenucleic acid encoding all or part of a xenogenous Ig gene is human.Preferably, the nucleic acid encodes a xenogenous antibody, such as ahuman antibody or a polyclonal antibody. In various embodiments, the Igchain or antibody is expressed in serum and/or milk.

In another aspect, the invention features a transgenic ungulate (e.g., abovine) having a mutation that reduces the expression of an endogenousantibody. Preferably, the mutation reduces the expression of functionalIgM heavy chain or substantially eliminates the expression of functionalIgM heavy chain. In some embodiments, a transcription terminationsequence is inserted in an endogenous mu heavy chain nucleic acid (e.g.,inserted downstream of the initial ATG codon in exon 2). In otherpreferred embodiments, the mutation reduces the expression of functionalIg light chain or substantially eliminates the expression of functionalIg light chain. In yet other preferred embodiments, the mutation reducesthe expression of functional IgM heavy chain and functional Ig lightchain, or the mutation substantially eliminates the expression offunctional IgM heavy chain and functional Ig light chain. In anotherpreferred embodiment, the ungulate has one or more nucleic acidsencoding all or part of a xenogenous Ig gene which undergoesrearrangement and expresses more than one xenogenous Ig molecule, suchas a xenogenous antibody protein.

Cells from transgenic ungulates expressing a xenogenous antibody and/orexpressing decreased levels of functional, endogenous antibody Theinvention also provides cells obtained from any of the ungulates (e.g.,bovines) of the invention or cells that are useful in the production ofany of the ungulates of the invention.

Accordingly, in another aspect, the invention features an ungulatesomatic cell (e.g., a bovine somatic cell) having one or more nucleicacids encoding all or part of a xenogenous Ig gene that is capable ofundergoing rearrangement and expressing one or more xenogenous Igmolecules in B cells. Preferably, the nucleic acid encoding all or partof a xenogenous Ig gene expresses a xenogenous antibody protein.Exemplary ungulate cells include fetal fibroblasts and B-cells.

In another aspect, the invention features an ungulate somatic cell(e.g., a bovine somatic cell) having a mutation in a nucleic acidencoding an Ig heavy and/or light chain. In preferred embodiments, thecell has a mutation in one or both alleles of the IgM heavy chain or theIg light chain. In some embodiments, a transcription terminationsequence is inserted in an endogenous mu heavy chain nucleic acid (e.g.,inserted downstream of the initial ATG codon in exon 2) or Ig lightchain nucleic acid. In some embodiments, a transcription terminationsequence is inserted in an endogenous mu heavy chain nucleic acid (e.g.,inserted downstream of the initial ATG codon in exon 2) or Ig lightchain nucleic acid. Exemplary mutations include nonsense and deletionmutations. In preferred embodiments, the cell also has one or morenucleic acids encoding all or part of a xenogenous Ig gene that iscapable of undergoing rearrangement and expressing one or morexenogenous Ig molecules in B cells. Preferably, the nucleic acidsencoding all or part of a xenogenous Ig gene expresses a xenogenousantibody, such as an antibody protein from another genus (e.g., a humanantibody). Exemplary ungulate cells include fetal fibroblasts andB-cells.

In another aspect, the invention features a hybridoma formed from thefusion of an ungulate B-cell of the invention with a myeloma cell.Preferably, the hybridoma secretes an exogenous antibody, such as ahuman antibody.

Methods for producing xenogenous antibodies in transgenic ungulates Theinvention also provides methods for producing antibodies using anungulate (e.g., a bovine embryo, fetus, calf, or adult) of theinvention. One such method involves administering one or more antigensof interest to an ungulate (e.g., a bovine embryo, fetus, calf, oradult) having one or more nucleic acids encoding a xenogenous antibodygene locus. The nucleic acid segments in the gene locus undergorearrangement resulting in the production of antibodies specific for theantigen, and the antibodies are recovered from the ungulate. Theantibodies may be monoclonal or polyclonal. Monoclonal and polyclonalantibodies against particular antigens have a variety of uses; forexample, they may be used as ingredients in prophylactic or therapeuticcompositions for infection of pathogenic microorganisms such as bacteriaor viruses. In various embodiments, the antibodies are recovered fromthe serum or milk of the ungulate. In preferred embodiments, theungulate has a mutation that reduces the expression of an endogenousantibody, that reduces the expression of functional IgM heavy chain, orthat reduces the expression of functional Ig light chain. In someembodiments, a transcription termination sequence is inserted in anendogenous mu heavy chain nucleic acid (e.g., inserted downstream of theinitial ATG codon in exon 2) or Ig light chain nucleic acid.

In a related aspect, the invention features another method of producingantibodies. This method involves recovering xenogenous antibodies froman ungulate (e.g., a bovine embryo, fetus, calf, or adult) havingnucleic acid encoding a xenogenous antibody gene locus. The nucleic acidsegments in the gene locus undergo rearrangement resulting in theproduction of xenogenous antibody proteins. In particular embodiments,the light chain of the antibodies and/or the heavy chain of theantibodies is encoded by a human nucleic acid. The antibodies may bemonoclonal or polyclonal. In particular embodiments, polyclonalantibodies, such as IgG antibodies generated without immunization of theungulate with a specific antigen, are used as a therapeutic substitutefor IVIG (intravenous immunoglobulin) produced from human serum. Invarious embodiments, the antibodies are recovered from the serum or milkof the ungulate. Preferably, the ungulate has a mutation that reducesthe expression of an endogenous antibody, reduces the expression offunctional IgM heavy chain, or reduces the expression of functional Iglight chain. Preferably, a transcription termination sequence isinserted in an endogenous mu heavy chain nucleic acid (e.g., inserteddownstream of the initial ATG codon in exon 2) or Ig light chain nucleicacid.

Methods for producing transgenic ungulates The invention also providesmethods for producing transgenic ungulates (e.g., bovine embryos,fetuses, calves, or adults). These methods may be used to producetransgenic ungulates having a desired mutation or having a desiredxenogenous nucleic acid.

In one such aspect, the invention features a method of producing atransgenic ungulate (e.g., bovine embryos, fetuses, calves, or adults)that involves inserting a cell, a chromatin mass from a cell, or anucleus from a cell into an oocyte. The cell includes a first mutationin an endogenous antibody heavy chain and/or light chain nucleic acid.The oocyte or an embryo formed from the oocyte is transferred into theuterus of a host ungulate, preferably under conditions that allow theoocyte or the embryo to develop into a fetus. Preferably, the fetusdevelops into a viable offspring. In some embodiments, the cell includesone or more nucleic acids encoding all or part of a xenogenous Ig genethat is capable of undergoing rearrangement and expressing one or morexenogenous Ig molecules in B cells. Preferably, a transcriptiontermination sequence is inserted in an endogenous mu heavy chain nucleicacid (e.g., inserted downstream of the initial ATG codon in exon 2) orIg light chain nucleic acid of the cell.

In various embodiments of the above aspect, the method also includesisolating a cell from the embryo, the fetus, or an offspring producedfrom the fetus and introducing a second mutation in an endogenousantibody heavy chain and/or light chain nucleic acid in the cell. Thecell, a chromatin mass from the cell, or a nucleus from the cell isinserted into an oocyte, and the oocyte or an embryo formed from theoocyte is transferred into the uterus of a host ungulate underconditions that allow the oocyte or the embryo to develop into a fetus.

In yet another aspect, the invention features another method ofproducing a transgenic ungulate (e.g., a bovine embryo, fetus, calf, oradult). This method involves inserting a cell having one or morexenogenous nucleic acids, a chromatin mass from the cell, or a nucleusfrom the cell into an oocyte. The xenogenous nucleic acid encodes all orpart of a xenogenous Ig gene, and the gene is capable of undergoingrearrangement and expressing more than one xenogenous Ig molecule in Bcells. The oocyte or an embryo formed from the oocyte is transferredinto the uterus of a host ungulate, preferably under conditions thatallow the oocyte or the embryo to develop into a fetus. Preferably, thefetus develops into a viable offspring. Preferably, the nucleic acidencoding all or part of a xenogenous Ig gene encodes a xenogenousantibody. In other preferred embodiments, the antibody is a polyclonalantibody. In yet other preferred embodiments, the immunoglobulin chainor antibody is expressed in serum and/or milk. In some embodiments, thedonor cell has a mutation in an endogenous antibody heavy chain and/orlight chain nucleic acid, such as an insertion of a transcriptiontermination sequence.

Methods for reducing the amount of undesired endogenous antibody in anungulate that expresses both endogenous and xenogenous antibody andmethods for producing xenogenous antibody in the ungulate The inventionalso features improved methods for producing primarily or onlyxenogenous antibody in a non-human mammal, such as an ungulate (e.g., abovine). In particular, these methods involve administering a compoundthat inhibits endogenous B-cell activity or that destroys endogenousB-cells, such as an anti-IgM or anti-Ig antibody, to a mammal thatexpresses both endogenous and xenogenous antibody in an amountsufficient to reduce the activity or amount of endogenous B-cells orantibody. These compounds may be administered during the normal periodof development of the mammal's immune system (i.e., during theembryonic, fetal, or postnatal stage) or after this period of immunesystem development. Preferably, antibodies that inhibit endogenousB-cells or antibodies do not substantially inhibit xenogenous B-cells orfully xenogenous antibodies. The resulting monoclonal or polyclonalxenogenous antibodies have a variety of uses; for example, they may beused as ingredients in prophylactic or therapeutic compositions forinfection of pathogenic microorganisms such as bacteria or viruses.

Accordingly, in one aspect, the invention provides a method of reducingthe quantity or activity of endogenous antibody in a non-human mammal(e.g., an ungulate). This method involves administering an antibody thatis reactive with a fully or partially endogenous antibody to an ungulate(e.g., a bovine) expressing both an endogenous antibody and a xenogenousantibody in an amount sufficient to reduce the quantity and/or activityof the fully or partially endogenous antibody. In a preferredembodiment, the antibody is administered prior to colostrum.

In a related aspect, the invention provides a method of producing axenogenous antibody in a non-human mammal (e.g., an ungulate). Thismethod involves administering an antibody that is reactive with a fullyor partially endogenous antibody to an ungulate (e.g., a bovine)expressing both an endogenous antibody and a xenogenous antibody in anamount sufficient to reduce the quantity or activity of the fully orpartially endogenous antibody. The xenogenous antibody is recovered fromthe ungulate. In preferred embodiments, the xenogenous antibody isrecovered from the serum or milk of the ungulate. In other preferredembodiment, some or all of the xenogenous antibodies are fullyxenogenous antibodies. In a preferred embodiment, the antibody isadministered prior to colostrum.

In another related aspect, the invention provides another method ofproducing a xenogenous antibody in a non-human mammal (e.g., anungulate). This method involves inserting a cell, nucleus, or chromatinmass into an enucleated oocyte, thereby forming a nuclear transferoocyte. The cell, nucleus, or chromatin mass has a nucleic acid encodinga first xenogenous antibody and a nucleic acid encoding a secondantibody reactive with an endogenous antibody. The nuclear transferoocyte or an embryo formed from the nuclear transfer oocyte istransferred to the uterus of a host mammal under conditions that allowit to develop into a fetus or live offspring. The fetus or offspringexpresses the xenogenous first antibody and the second antibody, and thesecond antibody reduces the quantity and/or activity of the endogenousantibody. Preferably, the second antibody is expressed under the controlof a liver-specific promoter. Some preferred second antibodies reactwith endogenous IgM or endogenous immunoglobulin molecules. Thexenogenous antibody is preferably recovered from the ungulate. Inpreferred embodiments, the xenogenous antibody is recovered from theserum or milk of the ungulate. In other preferred embodiment, some orall of the xenogenous antibodies are fully xenogenous antibodies

Methods for cloning non-human mammals using cells from two embryos inwhich cells from a nuclear transfer embryo are preferentiallyincorporated into the resulting fetal tissue and cells from anotherembryo are preferentially incorporated into the resulting placentaltissue to promote viability of the fetus The invention also providesimproved methods for cloning non-human mammals. These mammals are formedby combining cells from a nuclear transfer first embryo (e.g., an embryoformed by inserting a cell, nucleus, or chromatin mass into anenucleated oocyte) with cells from an in vitro fertilized,naturally-occurring, or parthenogenetically activated second embryo.This resulting chimeric embryo is transferred to the uterus of a hostmammal under conditions that allow it to develop into a fetus or liveoffspring. At least some of the cells from the second embryo arepreferably incorporated into placental tissue and promote the viabilityof the resulting chimeric embryo. Preferably, the majority of the cellsand their progeny from the nuclear transfer first embryo, rather thanfrom the second embryo, are incorporated into fetal tissue of theresulting chimeric embryo. Thus, the majority of the cells in the fetusor offspring from this chimeric embryo preferably have a genome that issubstantially identity or identical to that of the nuclear transferembryo first embryo rather than to that of the second embryo. To reducethe number of cells and their progeny from the second embryo that areincorporated into the fetal tissue or offspring, an antibody that isreactive with an antigen from the second embryo is administered to thechimeric embryo, fetus, or offspring in an amount sufficient to reducethe quantity and/or activity of cells from the second embryo that areincorporated into the fetus or offspring.

Accordingly, in one aspect the invention provides a method of cloning anon-human mammal. This method involves inserting a cell, nucleus, orchromatin mass into an oocyte, thereby forming a first embryo. One ormore cells from the first embryo are contacted with one or more cellsfrom a second embryo (e.g., an in vitro fertilized embryo,naturally-occurring embryo, or parthenogenetically activated embryo),thereby forming a third embryo. The third embryo is transferred into theuterus of a host mammal under conditions that allow the third embryo todevelop into a fetus or live offspring. An antibody that is reactivewith an antigen (e.g., a B-cell or germ cell antigen) from the secondembryo is administered to the third embryo, fetus, or offspring in anamount sufficient to reduce the quantity and/or activity of cells fromthe second embryo that are incorporated into the third embryo, fetus, oroffspring.

In a related aspect, the invention provides another method of cloning anon-human mammal. This method involves incubating a permeabilized cellin a reprogramming media under conditions that allow the removal of afactor from a nucleus, chromatin mass, or chromosome of thepermeabilized cell or the addition of a factor from the reprogrammingmedia to the nucleus, chromatin mass, or chromosome, thereby forming areprogrammed cell. The reprogrammed cell is inserted into an oocyte,thereby forming a first embryo. One or more cells from the first embryoare contacted with one or more cells from a second embryo (e.g., an invitro fertilized embryo, naturally-occurring embryo, orparthenogenetically activated embryo), thereby forming a third embryo.The third embryo is transferred into the uterus of a host mammal underconditions that allow the third embryo to develop into a fetus or liveoffspring. An antibody that is reactive with an antigen from the secondembryo is administered to the third embryo, fetus, or offspring in anamount sufficient to reduce the quantity and/or activity of cells fromthe second embryo that are incorporated into the third embryo, fetus, oroffspring.

The invention also provides methods for generating chimeric fetuses oroffspring in which cells from one of the initial embryos used to producethe chimeric fetus or offspring have a nucleic acid encoding axenogenous antibody (e.g., a human antibody). Additionally, cells fromthe aforementioned initial embryo or another initial embryo have anucleic acid encoding an antibody that is reactive with an endogenousantibody (e.g., an antibody naturally produced by cells from any of theinitial embryos used to generate the chimeric fetus or offspring) andthat reduces the amount or activity of an endogenous antibody in theresulting fetus or offspring.

In one such aspect, the invention features a method of cloning anon-human mammal. This method involves inserting a cell, nucleus, orchromatin mass into an oocyte, thereby forming a first embryo. The cell,nucleus, or chromatin mass has a nucleic acid encoding a xenogenousfirst antibody and a nucleic acid encoding a second antibody reactivewith an endogenous antibody. One or more cells from the first embryo arecontacted with one or more cells from a second embryo (e.g., an in vitrofertilized embryo, naturally-occurring embryo, or parthenogeneticallyactivated embryo), thereby forming a third embryo. The third embryo istransferred into the uterus of a host mammal under conditions that allowthe third embryo to develop into a fetus or live offspring. Theresulting fetus or offspring expresses the xenogenous first antibody andthe second antibody, and the second antibody reduces the quantity and/oractivity of an endogenous antibody. Preferably, the second antibody isexpressed under the control of a liver-specific promoter. Some preferredsecond antibodies react with endogenous IgM or endogenous immunoglobulinmolecules.

In a related aspect, the invention provides another method of cloning anon-human mammal. This method involves incubating a permeabilized cellin a reprogramming media under conditions that allow the removal of afactor from a nucleus, chromatin mass, or chromosome of thepermeabilized cell or the addition of a factor from the reprogrammingmedia to the nucleus, chromatin mass, or chromosome, thereby forming areprogrammed cell. The cell has a nucleic acid encoding a xenogenousfirst antibody and a nucleic acid encoding a second antibody reactivewith an endogenous antibody. The reprogrammed cell is inserted into anoocyte, thereby forming a first embryo. One or more cells from the firstembryo are contacted with one or more cells from a second embryo (e.g.,an in vitro fertilized embryo, naturally-occurring embryo, orparthenogenetically activated embryo), thereby forming a third embryo.The third embryo is transferred into the uterus of a host mammal underconditions that allow the third embryo to develop into a fetus or liveoffspring. The resulting fetus or offspring expresses the xenogenousfirst antibody and the second antibody, and the second antibody reducesthe quantity and/or activity of an endogenous antibody. Preferably, thesecond antibody is expressed under the control of a liver-specificpromoter. Some preferred second antibodies react with endogenous IgM orendogenous immunoglobulin molecules.

In preferred embodiments of any of the above cloning methods, the firstembryo comprises one or more nucleic acids encoding all or part of axenogenous immunoglobulin (Ig) gene which undergoes rearrangement andexpresses at least one xenogenous Ig molecule in B-cells. In otherpreferred embodiments, the first embryo comprises one or more nucleicacids encoding all or part of a rearranged xenogenous immunoglobulingene which expresses at least one xenogenous Ig molecule in B-cells. Insome embodiments, the first embryo or second embryo comprises a mutationthat reduces the expression of an endogenous antibody. In otherpreferred embodiments, the antibody is reactive with an antigenexpressed on the surface of B-cells or germ cells, such as an antibodyor a cell surface protein or receptor. In some embodiments, theadministered antibody is an anti-IgM or anti-immunoglobulin antibody. Ina preferred embodiment, the antibody is administered prior to colostrum.

Preferred ungulates for use in above methods Exemplary ungulates includemembers of the orders Perissodactyla and Artiodactyla, such as anymember of the genus Bos. Other preferred ungulates include sheep,big-horn sheep, goats, buffalos, antelopes, oxen, horses, donkeys, mule,deer, elk, caribou, water buffalo, camels, llama, alpaca, pigs, andelephants. In preferred embodiments, the recipient ungulate is less than50, 40, 30, 20, 10, 7, 5, 4, 3, 2, or 1 week old. In variousembodiments, the antibody is administered to a fetus during the first,second or third trimester. In yet other preferred embodiments, a fetusis allowed to develop until a chosen time in a pregnant or host mammal,and then the fetus is surgically removed or labor is induced usingstandard methods. For example, a viable fetus may be removed byCaesarian section, or labor may be artificially induced 1, 2, 3, 5, 10,15, 20, or more days prior to the normal term of the fetus. These youngrecipient ungulates may have a naturally suppressed immune system,thereby minimizing or preventing an adverse immune response to theadministered antibody which reduces endogenous antibodies. Otherpreferred ungulates naturally or spontaneously have an immune systemthat is less responsive than normal.

Preferred ungulates that express a xenogenous antibody contain naturallyarranged segments of human chromosomes (e.g., human chromosomalfragments) or artificial chromosomes that comprise artificiallyengineered human chromosome fragments (i.e., the fragments may berearranged relative to the human genome). Preferred ungulates have oneor more nucleic acids having a xenogenous antibody gene locus (e.g., anucleic acid encoding all or part of a xenogenous immunoglobulin (Ig)gene which undergoes rearrangement and expresses at least one xenogenousIg molecule). Preferably, the nucleic acid has unrearranged antibodylight chain nucleic acid segments in which all of the nucleic acidsegments encoding a V gene segment are separated from all of the nucleicacid segments encoding a J gene segment by one or more nucleotides.Other preferred nucleic acid have unrearranged antibody heavy chainnucleic acid segments in which either (i) all of the nucleic acidsegments encoding a V gene segment are separated from all of the nucleicacid segments encoding a D gene segment by one or more nucleotidesand/or (ii) all of the nucleic acid segments encoding a D gene segmentare separated from all of the nucleic acid segments encoding a J genesegment by one or more nucleotides.

Other preferred ungulates have one or more nucleic acids encoding all orpart of a rearranged xenogenous immunoglobulin (Ig) gene which expressesat least one xenogenous Ig molecule. In some embodiments, the nucleicacid is contained within a chromosome fragment. The nucleic acid may beintegrated into a chromosome of the ungulate or maintained in theungulate cell independently from the host chromosome.

In other preferred embodiments of any methods of the invention, thelight chain of the antibodies and/or the heavy chain of the xenogenousantibodies is encoded by a human nucleic acid. In preferred embodiments,the heavy chain is a mu heavy chain, and the light chain is a lambda orkappa light chain. In other preferred embodiments, the nucleic acidencoding the xenogenous immunoglobulin chain or antibody is in itsunrearranged form. In other preferred embodiments, more than one classof xenogenous antibody is produced by the ungulate. In variousembodiments, more than one different xenogenous Ig or antibody isproduced by the ungulate. The xenogenous antibody may be a polyclonal ormonoclonal antibody.

Preferred methods of generating ungulates for use in above methods Inparticular embodiments for the generation of transgenic ungulates thatexpress xenogenous antibodies, the ungulate is produced by inserting acell having one or more xenogenous nucleic acids into an oocyte. Thexenogenous nucleic acid encodes all or part of a xenogenous Ig gene, andthe gene is capable of undergoing rearrangement and expressing more thanone xenogenous Ig molecule in B-cells. The oocyte or an embryo formedfrom the oocyte is transferred into the uterus of a host ungulate underconditions that allow the oocyte or the embryo to develop into a fetus.Preferably, the fetus develops into a viable offspring. Preferably, thenucleic acid encoding all or part of a xenogenous Ig gene encodes axenogenous antibody. In other preferred embodiments, the antibody is apolyclonal antibody. In yet other preferred embodiments, theimmunoglobulin chain or antibody is expressed in serum and/or milk. Invarious embodiments, the nucleic acid is contained in a chromosomefragment, such as a ΔHAC or a ΔΔHAC. The nucleic acid can be maintainedin an ungulate cell independently from the host chromosome or integratedinto a chromosome of the cell. Preferably, the nucleic acid issubstantially human. In other embodiments, the xenogenous antibody is anantibody from another genus, such as a human antibody. Preferably, theungulate is a bovine, ovine, porcine, or caprine.

We have previously disclosed a variety of improved methods for cloningmammals that may be used to clone mammals for use in the methods of thepresent invention (U.S. Ser. No. 10/032,191, filed Dec. 21, 2001 andPCT/US01/50406, filed Dec. 21, 2001). In particular, these methodsinvolve the condensation of a donor nucleus into a chromatin mass toallow the release of nuclear components such as transcription factorsthat may promote the transcription of genes that are undesirable for thedevelopment of the nuclear transplant embryo into a viable offspring. Ina related method, a permeabilized cell is incubated with a reprogrammingmedia (e.g., a cell extract) to allow the addition or removal of factorsfrom the cell, and then the plasma membrane of the permeabilized cell isresealed to enclose the desired factors and restore the membraneintegrity of the cell. If desired, the steps of any of these methods maybe repeated one or more times or different reprogramming methods may beperformed sequentially to increase the extent of reprogramming,resulting in greater viability of the cloned fetuses.

In preferred embodiments that involve the use of these improved cloningmethods, the ungulate (e.g., bovine embryo, fetus, calf, or adult) isproduced using a method that involves (a) incubating a donor nucleus(e.g., a nucleus that has a nucleic acid encoding a xenogenous antibody)that preferably has less than four sets of homologous chromosomes (i.e.,has fewer than two pairs of complete chromatids) under conditions thatallow formation of a chromatin mass without causing DNA replication, (b)inserting the chromatin mass into an enucleated oocyte, thereby forminga nuclear transfer oocyte and (c) transferring the nuclear transferoocyte or an embryo formed from the nuclear transfer oocyte into theuterus of a host mammal, preferably under conditions that allow thenuclear transfer oocyte or embryo to develop into a fetus. In apreferred embodiment, the donor nucleus is incubated with areprogramming media (e.g., a cell extract) under conditions that allownuclear or cytoplasmic components such as transcription factors,repressor proteins, or chromatin remodeling proteins to be added to, orremoved from, the nucleus or resulting chromatin mass. Preferably, thedonor nucleus is contacted with one or more of the following underconditions that allow formation of a chromatin mass: a mitotic extractin the presence or absence of an anti-NuMA antibody, a detergent and/orsalt solution, or a protein kinase solution. In other preferredembodiments, the reconstituted oocyte or the resulting embryo expresseslamin A, lamin C, or NuMA protein at a level that is less than 5 foldgreater than the corresponding level expressed by a control oocyte or acontrol embryo with the same number of cells and from the same species.

In other preferred embodiments, the method for generating the ungulate(e.g., bovine embryo, fetus, calf, or adult) involves incubating apermeabilized cell (e.g., a cell that has a nucleic acid encoding axenogenous antibody) with a reprogramming media (e.g., a cell extract)under conditions that allow the removal of a factor (e.g., a nuclear orcytoplasmic component such as a transcription factor) from a nucleus,chromatin mass, or chromosome of the permeabilized cell or the additionof a factor to the nucleus, chromatin mass, or chromosome, therebyforming a reprogrammed cell. The reprogrammed cell is inserted into anenucleated oocyte, and the resulting oocyte or an embryo formed from theoocyte is transferred into the uterus of a host mammal, preferably underconditions that allow the oocyte or embryo to develop into a fetus. Inpreferred embodiments, the permeabilized cell is contacted with one ormore of the following under conditions that allow formation of achromatin mass: a mitotic extract in the presence or absence of ananti-NuMA antibody, a detergent and/or salt solution, or a proteinkinase solution. In yet another preferred embodiment, the permeabilizedcell is incubated with an interphase reprogramming media (e.g., aninterphase cell extract). In still another preferred embodiment, thenucleus in the permeabilized cell remains membrane-bounded, and thechromosomes in the nucleus do not condense during incubation with thisinterphase reprogramming media. In certain embodiments, incubating thepermeabilized cell in the reprogramming media does not cause DNAreplication or only causes DNA replication in less than 50, 40, 30, 20,10, or 5% of the cells. In other embodiments, incubating thepermeabilized cell in the reprogramming media causes DNA replication inat least 60, 70, 80, 90, 95, or 100% of the cells. In variousembodiments, the permeabilized cell is formed by incubating an intactcell with a protease such as trypsin, a detergent, such as digitonin, ora bacterial toxin, such as Streptolysin O. In a preferred embodiment,the reprogrammed cell is not incubated under conditions that allow themembrane of the reprogrammed cell to reseal prior to insertion into theoocyte. In yet another preferred embodiment, the reprogrammed cell isincubated under conditions that allow the membrane of the reprogrammedcell to reseal prior to insertion into the oocyte. In other preferredembodiments, the reconstituted oocyte or the resulting embryo expresseslamin A, lamin C, or NuMA protein at a level that is less than 5 foldgreater than the corresponding level expressed by a control oocyte or acontrol embryo with the same number of cells and from the same species.

Preferred Methods for Generating Chimeric Ungulates for Use in AboveMethods

Other preferred ungulates are chimeric ungulates or ungulates producedusing cells from two or more embryos. For example, cells from a nucleartransfer embryo (e.g., an embryo formed by inserting a cell, nucleus, orchromatin mass into an enucleated oocyte) can be combined with cellsfrom an in vitro fertilized, naturally-occurring, or parthenogeneticallyactivated embryo. Preferably, the majority of the cells and theirprogeny from the nuclear transfer embryo are incorporated into fetaltissue of the resulting chimeric embryo. At least some of the cells andtheir progeny from the second embryo are preferably incorporated intoplacental tissue and promote the viability of the resulting chimericembryo.

In preferred embodiments, the nuclear transfer embryo has a nucleic acidencoding a xenogenous antibody. Preferably, an antibody is administeredto the resulting embryo, fetus, or offspring that inhibits theendogenous B-cells or antibodies produced by cells derived from eitherinitial embryo but does not substantially inhibit the xenogenous B-cellsor antibodies.

Accordingly, in various preferred embodiments, the ungulate (e.g.,bovine embryo, fetus, calf, or adult) is produced by inserting a cell,nucleus, or chromatin mass (e.g., a cell, nucleus, or chromatin masshaving one or more nucleic acids encoding a xenogenous antibody) into anoocyte, thereby forming a first embryo. One or more cells from the firstembryo are contacted with one or more cells from a second embryo,thereby forming a third embryo. The second embryo is an in vitrofertilized embryo, naturally-occurring embryo, or parthenogeneticallyactivated embryo. The third embryo is transferred into the uterus of ahost mammal under conditions that allow the third embryo to develop intoa fetus.

In one embodiment, at least one of the first embryo and the secondembryo is a compaction embryo. In another embodiment, the first embryoand the second embryo are at different cell-stages. The first embryo andthe donor cell used to produce the second embryo can be from the samespecies or from different genuses or species. Preferably, at least 10,20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% cells in the trophectodermor placental tissue of the fetus are derived from the second embryo, orat least 30, 40, 50, 60, 70, 80, 90, 95, or 100% cells in the inner cellmass or fetal tissue of the fetus are derived from the first embryo. Inother preferred embodiments, the first embryo or the third embryoexpresses lamin A, lamin C, or NuMA protein at a level that is less than5 fold greater than the corresponding level expressed by a controlembryo with the same number of cells and from the same species.

In still other embodiments, the ungulate (e.g., bovine embryo, fetus,calf, or adult) is generated by contacting a donor nucleus (e.g., anucleus that encodes a xenogenous antibody) with a reprogramming media(e.g., cell extract) under conditions that allow formation of achromatin mass, and inserting the chromatin mass into an enucleatedoocyte, thereby forming a first embryo. One or more cells from the firstembryo are contacted with one or more cells from an in vitro fertilized,naturally-occurring, or parthenogenetically activated second embryo,forming a third embryo. The third embryo is transferred into the uterusof a host mammal under conditions that allow the third embryo to developinto a fetus. In a preferred embodiment, the chromatin mass is formed bycontacting a donor nucleus that has less than four sets of homologouschromosomes with a reprogramming media under conditions that allowformation of a chromatin mass without causing DNA replication.Preferably, the donor nucleus is contacted with one or more of thefollowing under conditions that allow formation of a chromatin mass: amitotic extract in the presence or absence of an anti-NuMA antibody, adetergent and/or salt solution, or a protein kinase solution.

In various embodiments, both the first embryo and the second embryo arecompaction embryos; both the first embryo and the second embryo areprecompaction embryos, or one of the embryos is a compaction embryo andthe other embryo is a precompaction embryo. The first embryo and thesecond embryo can be at different cell-stages or at the same cell-stage.The first embryo and the donor nucleus used to produce the second embryocan be from the same species or from different genuses or species.Preferably, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100%cells in the trophectoderm or placental tissue of the fetus are derivedfrom the second embryo, or at least 30, 40, 50, 60, 70, 80, 90, 95, or100% cells in the inner cell mass or fetal tissue of the fetus arederived from the first embryo. In other preferred embodiments, the firstembryo or the third embryo expresses lamin A, lamin C, or NuMA proteinat a level that is less than 5 fold greater than the corresponding levelexpressed by a control embryo with the same number of cells and from thesame species.

In another related aspect, the invention features yet another method ofcloning a mammal (e.g., bovine embryo, fetus, calf, or adult). Thismethod involves incubating a permeabilized cell (e.g., a cell that has anucleic acid encoding a xenogenous antibody) in a reprogramming media(e.g., cell extract) under conditions that allow the removal of a factorfrom a nucleus, chromatin mass, or chromosome of the permeabilized cellor the addition of a factor from the reprogramming media to the nucleus,chromatin mass, or chromosome, thereby forming a reprogrammed cell. Thereprogrammed cell is inserted into an enucleated oocyte, thereby forminga first embryo. One or more cells from the first embryo are contactedwith one or more cells from an in vitro fertilized, naturally-occurring,or parthenogenetically activated second embryo, forming a third embryo.The third embryo is transferred into the uterus of a host mammal underconditions that allow the third embryo to develop into a fetus. In apreferred embodiment, the permeabilized cell is incubated with areprogramming media (e.g., a cell extract) under conditions that allownuclear or cytoplasmic components such as transcription factors to beadded to, or removed from, the nucleus or resulting chromatin mass. Inother preferred embodiments, the permeabilized cell is contacted withone or more of the following under conditions that allow formation of achromatin mass: a mitotic extract in the presence or absence of ananti-NuMA antibody, a detergent and/or salt solution, or a proteinkinase solution. In yet another preferred embodiment, the permeabilizedcell is incubated with an interphase reprogramming media (e.g., aninterphase cell extract). In still another preferred embodiment, thenucleus in the permeabilized cell remains membrane-bounded, and thechromosomes in the nucleus do not condense during incubation with thisinterphase reprogramming media. In some embodiments, incubating thepermeabilized cell in the reprogramming media does not cause DNAreplication or only causes DNA replication in less than 50, 40, 30, 20,10, or 5% of the cells. In other embodiments, incubating thepermeabilized cell in the reprogramming media causes DNA replication inat least 60, 70, 80, 90, 95, or 100% of the cells. In variousembodiments, the permeabilized cell is formed by incubating an intactcell with a protease such as trypsin, a detergent, such as digitonin, ora bacterial toxin, such as Streptolysin O. In yet another preferredembodiment, the reprogrammed cell is incubated under conditions thatallow the membrane of the reprogrammed cell to reseal prior to insertioninto the oocyte. In various embodiments, both the first embryo and thesecond embryo are compaction embryos; both the first embryo and thesecond embryo are precompaction embryos, or one of the embryos is acompaction embryo and the other embryo is a precompaction embryo. Thefirst embryo and the second embryo can be at different cell-stages or atthe same cell-stage. The first embryo and the donor cell used to producethe second embryo can be from the same species or from different genusesor species. Preferably, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95,or 100% cells in the trophectoderm or placental tissue of the fetus arederived from the second embryo, or at least 30, 40, 50, 60, 70, 80, 90,95, or 100% cells in the inner cell mass or fetal tissue of the fetusare derived from the first embryo. In other preferred embodiments, thefirst embryo or the third embryo expresses lamin A, lamin C, or NuMAprotein at a level that is less than 5 fold greater than thecorresponding level expressed by a control embryo with the same numberof cells and from the same species.

In preferred embodiments of any of the above aspects involving ungulatesproduced using cells from two embryos, part or all of the zona pellucidaof the first embryo or second embryo is removed before the cells fromeach embryo are contacted. In one embodiment, the cells from the firstand second embryos are contacted by being placed adjacent to each otherin solution or on a solid support. In another embodiment, standardtechniques are used to inject cells from the first embryo into thesecond embryo. The cells can be injected into any region of the secondembryo, such as the periphery of the embryo between the zona pellucidaand the embryo itself. Exemplary naturally occurring embryos includeembryos that are surgically or nonsurgically removed from a pregnantmammal (e.g., a bovine) using standard methods. Exemplary in vitrofertilized embryos include intra-cytoplasmic sperm injection embryosgenerated using standard methods. It is also contemplated that cellsfrom more than two embryos (e.g., cells from 3, 4, 5, 6, or moreembryos) can be combined to form a chimeric embryo for generation of acloned mammal.

Preferred embodiments for generating ungulates for use in above methodsIn preferred embodiments of any of the above aspects, the reprogrammingmedia (e.g., a cell extract) is modified by the enrichment or depletionof a factor, such as a DNA methyltransferase, histone deacetylase,histone, protamine, nuclear lamin, transcription factor, activator, orrepressor. In other preferred embodiments, the level of expression ofNuMA or AKAP95 protein in the oocyte or chimeric embryo is at least 2,5, 10, or 20-fold greater in the nucleus than in the cytoplasm. In yetother embodiments, at least 30, 40, 50, 60, 70, 80, 90, or 100% of theAKAP95 protein in the oocyte or chimeric embryo is extracted with asolution of 0.1% Triton X-100, 1 mg/ml DNase I, and either 100 mM or 300mM NaCl. Preferably, the chromatin mass is purified from thereprogramming media (e.g., extract) prior to insertion into theenucleated oocyte. In another preferred embodiment, inserting thechromatin mass into the enucleated oocyte involves contacting thechromatin mass and the oocyte with a fusogenic compound under conditionsthat allow the chromatin mass to enter the oocyte. In yet anotherpreferred embodiment, the fetus develops into a viable offspring.Preferably, at least 1, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% ofthe nuclear transfer oocytes or embryos develop into viable offspring.In this method, the oocyte containing the chromatin mass or reprogrammedcell may be cultured under conditions that allow cell division and oneof the resulting cells may be recloned one or more times. The donornucleus, donor chromatin mass, or donor cell and the oocyte used in themethod may be from the same species, or they may be from differentspecies or genuses. The mammal may be a human or non-human mammal, andthe oocyte may be fertilized or unfertilized. Preferably the donornucleus, chromatin mass, or permeabilized cell is from a G₁ or G₀ phasecell. In addition, the genomic DNA of the cloned embryo, fetus, ormammal is preferably substantially identical to that of the donor cell.It is also contemplated that the chromatin mass or reprogrammed cell maybe inserted into an embryo for the production of a chimeric embryo,fetus, or mammal containing a mixture of cells with DNA substantiallyidentical to that of the chromatin mass or reprogrammed cell and cellswith DNA substantially identical to that of the naturally-occurringcells in the embryo. It is also contemplated that a nucleated oocyte maybe used in the methods of the invention.

The reprogramming media used in any of the aspects of the invention mayor may not contain exogenous nucleotides. In other preferredembodiments, a chromatin mass in a reprogramming media or formed in apermeabilized cell is contacted with a vector having a nucleic acidencoding a gene of interest under conditions that allow randomintegration or homologous recombination between the nucleic acid in thevector and the corresponding nucleic acid in the genome of the chromatinmass, resulting in the alteration of the genome of the chromatin mass.Due to the lack of an intact plasma membrane and the lack of a nuclearmembrane, a chromatin mass in a permeabilized cell or in solution may beeasier to genetically modify than a naturally-occurring cell. Examplesof cells that may be used to generate reprogramming extracts includeembryonic stem cells and adult stem cells from brain, blood, bonemarrow, pancreas, liver, skin, or any other organ or tissue. Otherexemplary reprogramming cell extracts include oocyte extracts (e.g.,bovine or sea urchin oocyte extracts) and male germ cell extracts (e.g.,spermatogonia, spermatocyte, spermatid, or sperm extracts fromvertebrates, invertebrates, or mammals such as bovine). The donor orpermeabilized cell can be non-immortalized or naturally, spontaneously,or genetically immortalized. The donor cell, permeabilized cell,recipient cell, or cytoplast can be from a source of any age, such as anembryo, fetus, youth, or adult mammal. Cells from younger sources mayhave acquired fewer spontaneous mutations and may have a longerlife-span after insertion into an oocyte.

Preferred ungulates with reduced levels of endogenous antibody for usein above methods The methods of the present invention may also be usedwith an ungulate (e.g., a bovine) that has a mutation that reduces theexpression of an endogenous antibody. Thus, less administered antibodyis required to eliminate this lower initial level of endogenousantibody. Preferably, the mutation reduces the expression of functionalIgM heavy chain or substantially eliminates the expression of functionalIgM heavy chain. In other preferred embodiments, the mutation reducesthe expression of functional Ig light chain or substantially eliminatesthe expression of functional Ig light chain. In yet other preferredembodiments, the mutation reduces the expression of functional IgM heavychain and functional Ig light chain, or the mutation substantiallyeliminates the expression of functional IgM heavy chain and functionalIg light chain. In some embodiments, a transcription terminationsequence is inserted in an endogenous mu heavy chain nucleic acid (e.g.,inserted downstream of the initial ATG codon in exon 2) or Ig lightchain nucleic acid. Preferably, the ungulate also has a mutation in oneor both alleles of an endogenous nucleic acid encodingalpha-(1,3)-galactosyltransferase, prion protein, and/or J chain. Inother preferred embodiments, the ungulate has a nucleic acid encoding anexogenous J chain, such as a human J chain. Preferably, the mutationreduces or eliminates the expression of the endogenousalpha-(1,3)-galactosyltransferase enzyme, galactosyl(α1,3)galactoseepitope, prion protein, and/or J chain. Preferably, the ungulateproduces human Igλ or IgM molecules containing human J chain.

Preferably, a transgenic ungulate with one or more mutations in anendogenous gene or genes is produced by inserting a cell, a chromatinmass from a cell, or a nucleus from a cell into an oocyte. The cell hasa first mutation in an endogenous gene that is not naturally expressedby the cell. The oocyte or an embryo formed from the oocyte istransferred into the uterus of a host ungulate under conditions thatallow the oocyte or the embryo to develop into a fetus. Preferably, thefetus develops into a viable offspring.

Preferred methods of generating ungulates with a mutation, such as amutation that leads to reduced levels of endogenous antibody, for use inthe above methods In other preferred embodiments, the first mutation isintroduced into the cell by inserting a nucleic acid comprising acassette which includes a promoter operably linked to a nucleic acidencoding a selectable marker and operably linked to one or more nucleicacids having substantial sequence identity to the endogenous gene to bemutated, whereby the cassette is integrated into one endogenous alleleof the gene. In other preferred embodiments, the mutation is introducedin the cell by inserting into the cell a nucleic acid comprising a firstcassette which includes a first promoter operably linked to a nucleicacid encoding a first selectable marker and operably linked to a firstnucleic acid having substantial sequence identity to the endogenous geneto be mutated, whereby the first cassette is integrated into a firstendogenous allele of the gene producing a first transgenic cell. Intothe first transgenic cell is inserted a nucleic acid comprising a secondcassette which includes a second promoter operably linked to a nucleicacid encoding a second selectable marker and operably linked to a secondnucleic acid having substantial sequence identity to the gene. Thesecond selectable marker differs from the first selectable marker, andthe second cassette is integrated into a second endogenous allele of thegene producing a second transgenic cell. In still other preferredembodiments, a cell is isolated from the embryo, the fetus, or anoffspring produced from the fetus, and another mutation is introducedinto a gene of the cell. A second round of nuclear transfer is thenperformed using the resulting cell, a chromatin mass from the cell, or anucleus from the cell to produce a transgenic ungulate with two or moremutations. The mutations are in the same or different alleles of a geneor are in different genes. The cell used in the first or optional secondround of nuclear transfer encodes a xenogenous antibody. In particularembodiments, the cell includes one or more nucleic acids encoding all orpart of a xenogenous Ig gene that is capable of undergoing rearrangementand expressing one or more xenogenous Ig molecules in B-cells. Inpreferred embodiments, the cell that is mutated is a fibroblast (e.g., afetal fibroblast). Preferably, the endogenous gene that is mutated isoperably linked to an endogenous promoter that is not active in afibroblast. In other preferred embodiments, the endogenous promoteroperably linked to the endogenous gene that is mutated is less than 80,70, 60, 50, 40, 30, 20, 10% as active as an endogenous promoter operablylinked to a endogenous housekeeping gene such as GAPDH. Promoteractivity may be measured using any standard assay, such as assays thatmeasure the level of mRNA or protein encoded by the gene (see, forexample, Ausubel et al. Current Protocols in Molecular Biology, volume2, p. 11.13.1-11.13.3, John Wiley & Sons, 1995). This method forgenerating a transgenic ungulate has the advantage of allowing a genethat is not expressed in the donor cell (i.e., the cell that is thesource of the genetic material used for nuclear transfer) to be mutated.

Preferably, the transgenic ungulate with a mutation in an endogenousantibody gene is produced by inserting a cell, a chromatin mass from acell, or a nucleus from a cell into an oocyte. The cell includes a firstmutation in an endogenous antibody heavy chain and/or light chainnucleic acid. The oocyte or an embryo formed from the oocyte istransferred into the uterus of a host ungulate under conditions thatallow the oocyte or the embryo to develop into a fetus. Preferably, thefetus develops into a viable offspring. Preferably, the cell used in theproduction of the transgenic ungulate has a mutation in one or bothalleles of an endogenous nucleic acid encodingalpha-(1,3)-galactosyltransferase, prion protein, and/or J chain. Inother preferred embodiments, the cell has a nucleic acid encoding anexogenous J chain, such as a human J chain. Preferably, the method forproducing the transgenic ungulate also includes isolating a cell fromthe embryo, the fetus, or an offspring produced from the fetus andintroducing a second mutation (e.g., an insertion of a transcriptiontermination sequence) in an endogenous antibody heavy chain and/or lightchain nucleic acid in the cell. The cell, a chromatin mass from thecell, or a nucleus from the cell is inserted into an oocyte, and theoocyte or an embryo formed from the oocyte is transferred into theuterus of a host ungulate under conditions that allow the oocyte or theembryo to develop into a fetus. The cell used in the first or optionalsecond round of nuclear transfer encodes a xenogenous antibody.

In other embodiments for the production of the above transgenicungulates, the cell used for generation of the transgenic ungulate isprepared by a method that includes inserting into the cell a nucleicacid having a cassette which includes a promoter operably linked to anucleic acid encoding a selectable marker and operably linked to one ormore nucleic acids having substantial sequence identity to the antibodyheavy chain or light chain nucleic acid. The cassette is integrated intoone endogenous allele of the antibody heavy chain or light chain nucleicacid.

In other embodiments, the cell is produced by inserting into the cell anucleic acid having a first cassette which includes a first promoteroperably linked to a nucleic acid encoding a first selectable marker andoperably linked to a first nucleic acid having substantial sequenceidentity to the antibody heavy chain or light chain nucleic acid. Thefirst cassette is integrated into a first endogenous allele of theantibody heavy chain or light chain nucleic acid producing a firsttransgenic cell. Into the first transgenic cell is inserted a nucleicacid having a second cassette which includes a second promoter operablylinked to a nucleic acid encoding a second selectable marker andoperably linked to a second nucleic acid having substantial sequenceidentity to the antibody heavy chain or light chain nucleic acid. Thesecond selectable marker differs from the first selectable marker. Thesecond cassette is integrated into a second endogenous allele of theantibody heavy chain or light chain nucleic acid producing a secondtransgenic cell.

In various embodiments of the invention, the nucleic acid used to mutatean endogenous ungulate nucleic acid (e.g., a knockout cassette whichincludes a promoter operably linked to a nucleic acid encoding aselectable marker and operably linked to a nucleic acid havingsubstantial sequence identity to the gene to be mutated) is notcontained in a viral vector, such as an adenoviral vector or anadeno-associated viral vector. For example, the nucleic acid may becontained in a plasmid or artificial chromosome that is inserted into anungulate cell, using a standard method such as transfection orlipofection that does not involve viral infection of the cell. In yetanother embodiment, the nucleic acid used to mutate an endogenousungulate nucleic acid (e.g., a knockout cassette which includes apromoter operably linked to a nucleic acid encoding a selectable markerand operably linked to a nucleic acid having substantial sequenceidentity to the gene to be mutated) is contained in a viral vector, suchas an adenoviral vector or an adeno-associated viral vector. Accordingto this embodiment, a virus containing the viral vector is used toinfect an ungulate cell, resulting in the insertion of a portion or theentire viral vector into the ungulate cell.

Preferred administered antibodies for use in above methods to eliminateundesired endogenous antibodies Preferred administered antibodiesinclude anti-IgM antibodies and antibodies reactive with a polyclonalmixture of endogenous ungulate antibodies. The administered antibody maybe monoclonal or polyclonal. In some embodiments, the administeredantibody is a bifunctional antibody, a fragment of an antibody, or amodified antibody. In certain embodiments, the administered antibody iscovalently linked to a toxin (e.g., diphtheria toxin, maytansinoids,CC-1065, anthracycline, or taxane), or a radiolabel. In someembodiments, the antibody is administered intravenously to the ungulate.Preferably, at least 0.25, 0.5, 1.0, 1.5, 2, 10, 20, or 50 grams of theantibody is administered in one or multiple doses to the ungulate. Inother embodiments, between 1 and 10 mg, 10 and 25 mg, 25 and 50 mg, 10and 100 mg, 50 and 100 mg, or 100 to 500 mg of the antibody isadministered in one or multiple doses to an ungulate fetus. If desired,the antibody may be administered in a pharmaceutically acceptablediluent, carrier, or excipient such as saline, buffered saline,dextrose, water, glycerol, ethanol, or a combination thereof.

In preferred embodiments, the administered antibody originates from anungulate of a different genus or species as the recipient ungulate. Inother preferred embodiments, the antibody is a bifunctional antibody.Still other preferred antibodies include those having, or consisting of,a ScFv, Fab, or F(ab′)₂ fragment. Other examples of preferred antibodiesinclude derivatized antibodies encoded by a fusion nucleic acid that hasbeen modified through gene fusion technology so that the nucleic acidencoding the antibody or a fragment of the antibody is operably linkedto a nucleic acid encoding a toxin or affinity tag. The covalentlylinked group in the derivatized antibody many be attached to theamino-terminus, carboxy-terminus, or between the amino- andcarboxy-termini, of the antibody or antibody fragment. By “affinity tag”is meant a peptide, protein, or compound that binds another peptide,protein, or compound. In a preferred embodiment, the affinity tag isused for purification or immobilization of the derivatized antibody. Inanother preferred embodiment, the affinity tag or toxin is used totarget the antibody to a specific cell, tissue, or organ system in vivo.

Preferred embodiments of above aspects Preferably, a transgenic cell orungulate of the invention has an insertion of a positive selectionmarker (e.g., an antibiotic resistance gene) into an endogenous nucleicacid encoding an immunoglobulin, alpha-(1,3)-galactosyltransferase,prion protein, or J chain. Desirably, the positive selection marker isoperably linked to a xenogenous promoter. In some embodiments, eachallele has an insertion of the same antibiotic resistance gene or adifferent antibiotic resistance gene. Preferably, a transcriptiontermination sequence is inserted into an endogenous nucleic acidencoding an immunoglobulin, alpha-(1,3)-galactosyltransferase, prionprotein, or J chain. For example, the transcription termination sequencemay be inserted downstream of the initial ATG codon in exon 2 of anendogenous mu heavy chain nucleic acid. In preferred embodiments, thecell or ungulate has one or more nucleic acids comprising one or moretransgenes and expressing an mRNA or protein encoded by thetransgene(s).

Preferably, the ungulate antiserum or milk has polyclonal humanimmunoglobulins. Preferably, the antiserum or milk is from a bovine,ovine, porcine, or caprine. In another preferred embodiment, the Igs aredirected against a desired antigen. In preferred embodiments, theantiserum is used as intravenous immunoglobulin (IVIG) for the treatmentor prevention of disease in humans. In another preferred embodiment, anantigen of interest is administered to the ungulate and Igs directedagainst the antigen are produced by the ungulate. Preferably, thenucleic acid segments in the xenogenous immunoglobulin gene locusrearrange, and xenogenous antibodies reactive with the antigen ofinterest are produced. Preferably, the antiserum and/or milk contains atleast 2, 5, 10, 20, or 50 fold more fully xenogenous antibody than fullyor partially endogenous antibody, or contains no fully or partiallyendogenous antibody. If desired, hybridomas and monoclonal antibodiescan be produced using xenogenous B-cells derived from theabove-described transgenic ungulates (for example, transgenic bovines).It is also contemplated that xenogenous antibodies (e.g., humanantibodies) isolated from ungulates may be subsequently chemicallymodified so that they are covalently linked to a toxin, therapeuticallyactive compound, enzyme, cytokine, radiolabel, fluorescent label, oraffinity tag. If desired, the fluorescent or radiolabel may be used forimaging of the antibody in vitro or in vivo.

In preferred embodiments of any of the methods of the invention, theungulate used in the method has a subpopulation of B-cells that expressfully xenogenous antibody (i.e., expresses antibody with fullyxenogenous heavy and light chains). Preferably, the amount ofendogenous, functional antibody is decreased by at least 10, 25, 50, 75,90, 95, or 100%, and/or the amount of xenogenous, functional antibody isdecreased by less than 75, 50, 25, or 10%. In other preferredembodiments, the decrease in the amount of endogenous, functionalantibody is at least 2, 5, 10, 20, 30, or 50-fold greater than thedecrease in the amount of xenogenous, functional antibody. In anotherpreferred embodiment, endogenous antibody is substantially eliminatedfrom the ungulate. Preferably, B-cells that express only endogenousantibody or that express both endogenous and xenogenous antibodymolecules (e.g., heavy and light chains) are substantially eliminatedfrom the ungulate. In other preferred embodiments, the number and/oractivity of endogenous B-cells expressing endogenous antibody isinhibited by at least 25, 50, 75, 90, or 95%. Preferably, the antibodyis administered to the ungulate during or after the normal period ofimmune system development of the ungulate. For example, the antibody maybe administered during the fetal, embryonic, or postnatal stage of therecipient ungulate.

Preferred donor cells for use in generating ungulates used in the abovemethods Examples of preferred donor cells include differentiated cellssuch as epithelial cells, neural cells, epidermal cells, keratinocytes,hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes,T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, andmuscle cells; and undifferentiated cells such as embryonic cells (e.g.,stem cells and embryonic germ cells). In another preferred embodiment,the cell is from the female reproductive system, such as a mammarygland, ovarian cumulus, granulosa, or oviductal cell. Other preferredcells include fetal cells and placental cells. Preferred cells alsoinclude those from any organ, such as the bladder, brain, esophagus,fallopian tube, heart, intestines, gallbladder, kidney, liver, lung,ovaries, pancreas, prostate, spinal cord, spleen, stomach, testes,thymus, thyroid, trachea, ureter, urethra, and uterus. In yet anotherpreferred embodiment, the nucleus, permeabilized cell, or chromosomesare from a transgenic cell or mammal or contain a mutation not found inthe donor cell or not found in a naturally-occurring cell.

Preferred transgenic donor nuclei and donor cells encode proteins thatconfer improved resistance to disease or parasites in the cloned mammal.Alternatively, the donor nuclei or donor cells may be engineered so thatthe cloned mammal produces a recombinant product, such as the productionof a human protein in the urine, blood, or milk of a bovine. Forexample, proteins may be expressed in the urine of cattle by inserting apolynucleotide sequence encoding a human protein under the control of anuroplakin promoter. Examples of therapeutic proteins that may beproduced in the milk of cloned bovines include human clotting factorssuch as any of factors I to XIII (Voet and Voet, Biochemistry, JohnWiley & Sons, New York, 1990). These heterologous proteins may beexpressed under the control of a prolactin promoter or any otherpromoter suitable for expression in the milk of a bovine. Recombinantproteins from these or other tissues or fluids may be purified usingstandard purification methods (see, for example, Ausubel et al., supra).

Cell permeabilization methods In another aspect, the invention featuresa method of permeabilizing a cell or a population of cells. This methodinvolves incubating one or more cells with one or more proteases (e.g.,trypsin) under conditions that allow the permeabilization of the cell(e.g., the permeabilization of the plasma membrane). Preferredconcentrations of protease include between 0.1 and 10 mg/ml protease,such as between 0.1 and 1 mg/ml, 1 and 5 mg/ml, and 5 and 10 mg/mlprotease. In some embodiments, electroporation, digitonin, saponin,and/or mechanical shear is used. Examples of cells that can bepermeabilized using this method include germ, somatic, embryonic, fetal,adult, differentiated, and undifferentiated cells. Other exemplary cellsinclude any of the cells listed in the above section as preferred donorcells. In some embodiments, the cells are incubated with the proteasefor at least 1, 5, 10, 30, or 60 minutes or between 1 and 60 minutes(e.g., between 1 and 10 minutes). In some embodiments, the permeabilizedcells are placed in a reprogramming media (e.g., a mitotic or interphaseextract) after permeabilization. In preferred embodiments, the cells areresealed after incubation in the extract and used in one of the cloningmethods described herein.

Definitions As used herein, by “artificial chromosome” is meant amammalian chromosome or fragment thereof which has an artificialmodification such as the addition of a selectable marker, the additionof a cloning site, the deletion of one or more nucleotides, thesubstitution of one or more nucleotides, and the like. By “humanartificial chromosome (HAC)” is meant an artificial chromosome generatedfrom one or more human chromosome(s). An artificial chromosome can bemaintained in the host cell independently from the endogenouschromosomes of the host cell. In this case, the HAC can stably replicateand segregate along side endogenous chromosomes. Alternatively, it maybe translocated to, or inserted into, an endogenous chromosome of thehost cell. Two or more artificial chromosomes can be introduced to thehost cell simultaneously or sequentially. For example, artificialchromosomes derived from human chromosome #14 (comprising the Ig heavychain gene), human chromosome #2 (comprising the Ig kappa chain gene),and human chromosome #22 (comprising the Ig lambda chain gene) can beintroduced. Alternatively, an artificial chromosome(s) comprising both axenogenous Ig heavy chain gene and Ig light chain gene, such as ΔHAC orΔΔHAC, may be introduced. Preferably, the heavy chain loci and the lightchain loci are on different chromosome arms (i.e., on different side ofthe centromere). In still other preferred embodiments, the total size ofthe HAC is less than or equal to approximately 10, 9, 8, or 7 megabases.

By “a nucleic acid in its pre-arranged or unrearranged form” is meant anucleic acid that has not undergone V(D)J recombination. In preferredembodiments, all of the nucleic acid segments encoding a V gene segmentof an antibody light chain are separated from all of the nucleic acidsegments encoding a J gene segment by one or more nucleotides.Preferably, all of the nucleic acid segments encoding a V gene segmentof an antibody heavy chain are separated from all of the nucleic acidsegments encoding a D gene segment by one or more nucleotides, and/orall of the nucleic acid segments encoding a D gene segment of anantibody heavy chain are separated from all of the nucleic acid segmentsencoding a J gene segment by one or more nucleotides. Preferably, anucleic acid in its unrearranged form is substantially human. In otherpreferred embodiments, the nucleic acid is at least 70, 80, 90, 95, or99% identical to the corresponding region of a naturally-occurringnucleic acid from a human.

By “chromatin mass” is meant more than one chromosome not enclosed by amembrane. Preferably, the chromatin mass contains all of the chromosomesof a cell. An artificially induced chromatin mass containing condensedchromosomes may be formed by exposure of a nucleus to a mitoticreprogramming media (e.g., a mitotic extract) as described herein.Alternatively, an artificially induced chromatin mass containingdecondensed or partially condensed chromosomes may be generated byexposure of a nucleus to one of the following, as described herein: amitotic extract containing an anti-NuMA antibody, a detergent and/orsalt solution, or a protein kinase solution. A chromatin mass maycontain discrete chromosomes that are not physically touching each otheror may contain two or more chromosomes that are in physical contact.

If desired, the level of chromosome condensation may be determined usingstandard methods by measuring the intensity of staining with the DNAstain, DAPI. As chromosomes condense, this staining intensity increases.Thus, the staining intensity of the chromosomes may be compared to thestaining intensity for decondensed chromosomes in interphase (designated0% condensed) and maximally condensed chromosomes in mitosis (designated100% condensed). Based on this comparison, the percent of maximalcondensation may be determined. Preferred condensed chromatin masses areat least 50, 60, 70, 80, 90, or 100% condensed. Preferred decondensed orpartially condensed chromatin masses are less than 50, 40, 30, 20, or10% condensed.

By “nucleus” is meant a membrane-bounded organelle containing most orall of the DNA of a cell. The DNA is packaged into chromosomes in adecondensed form. Preferably, the membrane encapsulating the DNAincludes one or two lipid bilayers or has nucleoporins.

By “nucleus that has less than four sets of homologous chromosomes” ismeant a nucleus that has a DNA content of less than 4n, where “n” is thenumber of chromosomes found in the normal haploid chromosome set of amammal of a particular genus or species. Such a nucleus does not havefour copies of each gene or genetic locus. Preferably, the nucleus isdiploid and thus has two sets of homologous chromosomes but has lessthan two complete pairs of chromatids.

By “pronucleus” is meant a haploid nucleus resulting from meiosis or anuclear transfer pronucleus. The female pronucleus is the nucleus of theoocyte or ovum before fusion with the male pronucleus. The malepronucleus is the sperm nucleus after it has entered the oocyte or ovumat fertilization but before fusion with the female pronucleus. A nucleartransfer pronucleus is a pronucleus (e.g., a diploid pronucleus) thatforms after introduction of a donor cell, nucleus, or chromatin massinto an oocyte. The nuclear transfer pronucleus has less than four setsof homologous chromosomes.

By “donor cell” is meant a cell from which a nucleus or chromatin massis derived, or a permeabilized cell.

By “permeabilization” is meant the formation of pores in the plasmamembrane or the partial or complete removal of the plasma membrane.

By “reprogramming media” is meant a solution that allows the removal ofa factor from a cell, nucleus, chromatin mass, or chromosome or theaddition of a factor from the solution to the cell, nucleus, chromatinmass, or chromosome. Preferably, the addition or removal of a factorincreases or decreases the level of expression of an mRNA or protein inthe donor cell, chromatin mass, or nucleus or in a cell containing thereprogrammed chromatin mass or nucleus. In another embodiment,incubating a permeabilized cell, chromatin mass, or nucleus in thereprogramming media alters a phenotype of the permeabilized cell or acell containing the reprogrammed chromatin mass or nucleus relative tothe phenotype of the donor cell. In yet another embodiment, incubating apermeabilized cell, chromatin mass, or nucleus in the reprogrammingmedia causes the permeabilized cell or a cell containing thereprogrammed chromatin mass or nucleus to gain or lose an activityrelative to the donor cell.

Exemplary reprogramming media include solutions, such as buffers, thatdo not contain biological molecules such as proteins or nucleic acids.Such solutions are useful for the removal of one or more factors from anucleus, chromatin mass, or chromosome. Other preferred reprogrammingmedias are extracts, such as cellular extracts from cell nuclei, cellcytoplasm, or a combination thereof. Exemplary cell extracts includeextracts from oocytes (e.g., mammalian, vertebrate, or invertebrateoocytes), male germ cells (mammalian, vertebrate, or invertebrate germcells such as spermatogonia, spermatocyte, spermatid, or sperm), andstem cells (e.g., adult or embryonic stem cells). Yet otherreprogramming media are solutions or extracts to which one or morenaturally-occurring or recombinant factors (e.g., nucleic acids orproteins such as DNA methyltransferases, histone deacetylases, histones,protamines, nuclear lamins, transcription factors, activators,repressors, chromatin remodeling proteins, growth factors, interleukins,cytokines, or other hormones) have been added, or extracts from whichone or more factors have been removed. Still other reprogramming mediainclude solutions of detergent (e.g., 0.01% to 0.1%, 0.1% to 0.5%, or0.5% to 2% ionic or non-ionic detergent such as one or more of thefollowing detergents: SDS, Triton X-100, Triton X-114, CHAPS,Na-deoxycholate, n-octyl glucoside, Nonidet P40, IGEPAL, Tween 20, Tween40, or Tween 80), salt (e.g., ˜0.1, 0.15, 0.25, 0.5, 0.75, 1, 1.5, or 2M NaCl or KCl), polyamine (e.g., ˜1 μM, 10 μM, 100 μM, 1 mM or 10 mMspermine, spermidine, protamine, or poly-L-lysine), a protein kinase(e.g., cyclin-dependent kinase 1, protein kinase C, protein kinase A,MAP kinase, calcium/calmodulin-dependent kinase, CK1 casein kinase, orCK2 casein kinase), and/or a phosphatase inhibitor (e.g., ˜10 μM, 100μM, 1 mM, 10 mM, 50 mM, 100 mM of one or more of the followinginhibitors: Na-orthovanadate, Na-pyrophosphate, Na-fluoride, NIPP1,inhibitor 2, PNUTS, SDS22, AKAP149, or ocadaic acid). In someembodiments, the reprogramming medium contains an anti-NuMA antibody. Ifdesired, multiple reprogramming media may be used simultaneously orsequentially to reprogram a donor cell, nucleus, or chromatin mass.

By “interphase reprogramming media” is meant a media (e.g., aninterphase cell extract) that induces chromatin decondensation andnuclear envelope formation.

By “mitotic reprogramming media” is meant a media (e.g., a mitotic cellextract) that induces chromatin condensation and nuclear envelopebreakdown.

By “reprogrammed cell” is meant a cell that has been exposed to areprogramming media. Preferably, at least 1, 5, 10, 15, 20, 25, 50, 75,100, 150, 200, 300, or more mRNA or protein molecules are expressed inthe reprogrammed cell that are not expressed in the donor orpermeabilized cell. In another preferred embodiment, the number of mRNAor protein molecules that are expressed in the reprogrammed cell, butnot expressed in the donor or permeabilized cell, is between 1 and 5, 5and 10, 10 and 25, 25 and 50, 50 and 75, 75 and 100, 100 and 150, 150and 200, or 200 and 300, inclusive. Preferably, at least 1, 5, 10, 15,20, 25, 50, 75, 100, 150, 200, 300, or more mRNA or protein moleculesare expressed in the donor or permeabilized cell that are not expressedin the reprogrammed cell. In yet another preferred embodiment, thenumber of mRNA or protein molecules that are expressed in the donor orpermeabilized cell, but not expressed in the reprogrammed cell, isbetween 1 and 5, 5 and 10, 10 and 25, 25 and 50, 50 and 75, 75 and 100,100 and 150, 150 and 200, or 200 and 300, inclusive. In still anotherpreferred embodiment, these mRNA or protein molecules are expressed inboth the donor cell (i.e., the donor or permeabilized starting cell) andthe reprogrammed cell, but the expression levels in these cells differby at least 2, 5, 10, or 20-fold, as measured using standard assays(see, for example, Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, 2000).

By “addition of a factor” is meant the binding of a factor to chromatin,a chromosome, or a component of the nuclear envelope, such as thenuclear membrane or nuclear matrix. Alternatively, the factor isimported into the nucleus so that it is bounded or encapsulated by thenuclear envelope. Preferably, the amount of factor that is bound to achromosome or located in the nucleus increases by at least 25, 50, 75,100, 200, or 500%.

By “removal of a factor” is meant the dissociation of a factor fromchromatin, a chromosome, or a component of the nuclear envelope, such asthe nuclear membrane or nuclear matrix. Alternatively, the factor isexported out of the nucleus so that it is no longer bounded orencapsulated by the nuclear envelope. Preferably, the amount of factorthat is bound to a chromosome or located in the nucleus decreases by atleast 25, 50, 75, 100, 200, or 500%.

By “enrichment or depletion of a factor” is meant the addition orremoval of a naturally-occurring or recombinant factor by at least 20,40, 60, 80, or 100% of the amount of the factor originally present in anreprogramming media (e.g., a cell extract). Alternatively, anaturally-occurring or recombinant factor that is not naturally presentin the reprogramming media may be added. Preferred factors includeproteins such as DNA methyltransferases, histone deacetylases, histones,protamines, nuclear lamins, transcription factors, activators, andrepressors; membrane vesicles, and organelles. In one preferredembodiment, the factor is purified prior to being added to thereprogramming media, as described below. Alternatively, one of thepurification methods described below may be used to remove an undesiredfactor from the reprogramming media.

By “recloned” is meant used in a second round of cloning. In particular,a cell from an embryo, fetus, or adult generated from the methods of theinvention may be incubated in a mitotic reprogramming media (e.g., amitotic cell extract) to form a chromatin mass for insertion into anenucleated oocyte, as described above. Alternatively, the cell may bepermeabilized, incubated in a reprogramming media, and inserted into anenucleated oocyte, as described above. Performing two or more rounds ofcloning may result in additional reprogramming of the donor chromatinmass or donor cell, thereby increasing the chance of generating a viableoffspring after the last round of cloning.

By “nuclear transfer” is meant inserting a nucleus or a cell containinga nucleus into an oocyte. Any appropriate method may be used for thisinsertion into an oocyte, such as microinjection, electroporation, orcell fusion. In some embodiments, the nucleus is formed from a chromatinmass or a cell containing a chromatin mass, as described herein.

By “chromatin transfer” is meant inserting a chromatin mass or a cellcontaining a chromatin mass into an oocyte. Any appropriate method maybe used for this insertion into an oocyte, such as microinjection,electroporation, or cell fusion. In some embodiments, the chromatin massis formed by incubating a nucleus or a cell containing a nucleus in areprogramming media, as described herein.

By “viable offspring” is meant a mammal that survives ex utero.Preferably, the mammal is alive for at least one second, one minute, onehour, one day, one week, one month, six months, or one year from thetime it exits the maternal host. The mammal does not require thecirculatory system of an in utero environment for survival.

By “nuclear transfer oocyte” or “nuclear transplant oocyte” is meant anoocyte in which a donor cell, nucleus, or chromatin mass is inserted orfused. An embryo formed from the oocyte is referred to as a “nucleartransfer” or “nuclear transplant” embryo.

By “embryo” or “embryonic” is meant a developing cell mass that has notimplanted into the uterine membrane of a maternal host. Hence, the term“embryo” may refer to a fertilized oocyte; an oocyte containing a donorchromatin mass, nucleus, or reprogrammed cell; a pre-blastocyst stagedeveloping cell mass; or any other developing cell mass that is at astage of development prior to implantation into the uterine membrane ofa maternal host and prior to formation of a genital ridge. An embryo mayrepresent multiple stages of cell development. For example, a one cellembryo can be referred to as a zygote; a solid spherical mass of cellsresulting from a cleaved embryo can be referred to as a morula, and anembryo having a blastocoel can be referred to as a blastocyst. An“embryonic cell” is a cell isolated from or contained in an embryo.

By “cells derived from an embryo” is meant cells that result from thecell division of cells in the embryo.

By “chimeric embryo” is meant an embryo formed from cells from two ormore embryos. The resulting fetus or offspring can have cells that arederived from only one of the initial embryos or cells derived from morethan one of the initial embryos. If desired, the percentage of cellsfrom each embryo that are incorporated into the placental tissue andinto the fetal tissue can be determined using standard FISH analysis oranalysis of a membrane dye added to one embryo.

By “chimeric ungulate” is meant an ungulate formed from cells from twoor more embryos. The ungulate can have cells that are derived from onlyone of the initial embryos or cells derived from more than one of theinitial embryos. If desired, the percentage of cells from each embryothat are incorporated into the placental tissue and into the fetaltissue can be determined using standard FISH analysis or analysis of amembrane dye added to one embryo.

By “precompaction embryo” is meant an embryo prior to compaction. Aprecompaction embryo expresses essentially no E-cadherin on the surfaceof its blastomeres. Preferred precompaction embryos express at least 3,5, 10, 20, 30, or 40-fold less E-cadherin than a fully compacted embryoof the same species, or express no E-cadherin.

By “compaction embryo” is meant an embryo undergoing compaction orfollowing compaction. The blastomeres of a compaction embryo expressE-cadherin on their surface. This E-cadherin expression can be measuringusing standard methods with an anti-E-cadherin antibody. E-cadherinincreases the adherence between blastomeres. Preferred compactionembryos include embryos in which the compaction process is completed.Other preferred compaction embryos express at least 3, 5, 10, 20, 30, or40-fold more E-cadherin than a precompaction embryo of the same species.

By “fetus” is meant a developing cell mass that has implanted into theuterine membrane of a maternal host. A fetus may have defining featuressuch as a genital ridge which is easily identified by a person ofordinary skill in the art. A “fetal cell” is any cell isolated from orcontained in a fetus.

By “parthenogenesis” or “parthenogenetic activation” is meantdevelopment of an oocyte or ovum without fusion of its nucleus with amale pronucleus to form a zygote. For example, an oocyte can be inducedto divide without fertilization.

By “zona pellucida” is meant a translucent, elastic, noncellular layersurrounding the oocyte or ovum of many mammals.

By “trophectoderm” is meant the outermost layer of cells surrounding theblastocoel during the blastocyst stage of mammalian embryonicdevelopment. Trophectoderm gives rise to most or all of the placentaltissue upon further development.

By “inner cell mass” is meant the cells surrounded by the trophectoderm.The inner cell mass cells give rise to most of the fetal tissues uponfurther development.

By “mRNA or protein specific for one cell type” is meant an mRNA orprotein that is expressed in one cell type at a level that is at least10, 20, 50, 75, or 100 fold greater than the expression level in allother cell types. Preferably, the mRNA or protein is only expressed inone cell type.

By “mutation” is meant an alteration in a naturally-occurring orreference nucleic acid sequence, such as an insertion, deletion,frameshift mutation, silent mutation, nonsense mutation, or missensemutation. Preferably, the amino acid sequence encoded by the nucleicacid sequence has at least one amino acid alteration from anaturally-occurring sequence. Examples of recombinant DNA techniques foraltering the genomic sequence of a cell, embryo, fetus, or mammalinclude inserting a DNA sequence from another organism (e.g., a human)into the genome, deleting one or more DNA sequences, and introducing oneor more base mutations (e.g., site-directed or random mutations) into atarget DNA sequence. Examples of methods for producing thesemodifications include retroviral insertion, artificial chromosometechniques, gene insertion, random insertion with tissue specificpromoters, homologous recombination, gene targeting, transposableelements, and any other method for introducing foreign DNA. All of thesetechniques are well known to those skilled in the art of molecularbiology (see, for example, Ausubel et al., supra). Chromatin masses,chromosomes, and nuclei from transgenic cells containing modified DNA ordonor transgenic cells may be used in the methods of the invention.

By “immortalized” is meant capable of undergoing at least 25, 50, 75,90, or 95% more cell divisions than a naturally-occurring control cellof the same cell type, genus, and species as the immortalized cell orthan the donor cell from which the immortalized cell was derived.Preferably, an immortalized cell is capable of undergoing at least 2, 5,10, or 20-fold more cell divisions than the control cell. Morepreferably, the immortalized cell is capable of undergoing an unlimitednumber of cell divisions. Examples of immortalized cells include cellsthat naturally acquire a mutation in vivo or in vitro that alters theirnormal growth-regulating process. Still other preferred immortalizedcells include cells that have been genetically modified to express anoncogene, such as ras, myc, abl, bcl2, or neu, or that have beeninfected with a transforming DNA or RNA virus, such as Epstein Barrvirus or SV40 virus (Kumar et al., Immunol. Lett. 65:153-159, 1999;Knight et al., Proc. Nat. Acad. Sci. USA 85:3130-3134, 1988; Shammah etal., J. Immunol. Methods 160-19-25, 1993; Gustafsson and Hinkula, Hum.Antibodies Hybridomas 5:98-104, 1994; Kataoka et al., Differentiation62:201-211, 1997; Chatelut et al., Scand. J. Immunol. 48:659-666, 1998).Cells can also be genetically modified to express the telomerase gene(Roques et al., Cancer Res. 61:8405-8507, 2001).

By “non-immortalized” is meant not immortalized as described above.

By “fusogenic compound” is meant a compound that increases theprobability that a chromatin mass or nucleus is inserted into arecipient cell when located adjacent to the cell. For example, thefusogenic compound may increase the affinity of a chromatin mass or anucleus for the plasma membrane of a cell. The fusogenic compound mayalso promote the joining of the nuclear membrane of a nucleus with theplasma membrane of a cell.

By “substantially identical” is meant having a sequence that is at least60, 70, 80, 90, or 100% identical to that of another sequence. Sequenceidentity is typically measured using sequence analysis software with thedefault parameters specified therein (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Thissoftware program matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.

By “reducing the quantity and/or activity of endogenous antibody” ismeant reducing the amount of endogenous antibodies produced by a B-cellor a population of B-cells. This reduction in the amount of endogenousantibodies may be due to a decrease in the amount of endogenousantibodies produced per B-cell, a decrease in the number of functionalendogenous B-cells, or a combination thereof. Preferably, the amount ofan endogenous antibody secreted by a B-cell or expressed on the surfaceof a B-cell expressing or secreting endogenous antibody is reduced by atleast 25, 50, 75, 90, or 95%. In another preferred embodiment, thenumber of endogenous B-cells in a sample from the recipient mammal, suchas a blood sample, is reduced by at least 25, 50, 75, 90, or 95%.

By “substantially eliminated” is meant a decrease of at least 75, 80,95, or 100%. In preferred embodiments, an ungulate in which B-cells thatexpress fully or partially endogenous antibody are substantiallyeliminated has an undetectable amount of these B-cells. In otherpreferred embodiments, an ungulate in which fully or partiallyendogenous antibodies are substantially eliminated has an undetectableamount of these antibodies.

By “mammal with an immune system that is less responsive than normal” ismeant a recipient mammal that naturally or spontaneously has an innateor adaptive immune system that is less active than normal. For example,the recipient mammal may have fewer B-cells, fewer Ig moleculesexpressed on the surface of B-cells, fewer antibody molecules secretedby B-cells, fewer T-cells, fewer cytokine molecules produced by T-cellsexposed to an antigen or mitogen, less cytotoxic activity orproliferation of T-cells in response to an antigen or mitogen, or fewerantibody or cytokine molecules produced in response to administration ofan antigen, based on standard methods such as those described herein.Preferably, the number of any of the above cells or immunoglobulins orthe level of any of the above activities in a recipient mammal is lessthan the average number of cells or immunoglobulins or the average levelof activity for mammals of the same genius, species, and age. In otherpreferred embodiments, the number of any of these cells orimmunoglobulins or the level of any of these activities in a recipientmammal is less 90, 80, 70, 60, 50, 40, 30, or 20% of the correspondingnumber of cells or immunoglobulins or the corresponding level ofactivity in another mammal of the same genus, species, and age. Any ofthese assays may also be used to identify the mammals in a population ofpotential recipient mammals with the least active immune systems.

By “fully endogenous antibody” is meant an antibody that has an aminoacid sequence that consists entirely of sequence endogenous to the hostorganism. If desired, the amount of fully endogenous antibody in asample from a transgenic ungulate may be measured by determining theamount of antibody in the sample that (i) reacts with an antibody (e.g.,anti-bovine immunoglobulin antibody) reactive with endogenous antibodybut (ii) does not react with an antibody (e.g., anti-humanimmunoglobulin antibody) reactive with xenogenous antibody. If desired,the antibody reactive with endogenous antibody (e.g., anti-bovineimmunoglobulin antibody) that is used in this assay is preabsorbedagainst xenogenous (e.g., human) immunoglobulin to ensure thatantibodies which cross react with xenogenous antibody are removed.Similarly, the antibody reactive with xenogenous antibody (e.g.,anti-human immunoglobulin antibody) that is used in this assay ispreabsorbed against endogenous (e.g., bovine) immunoglobulin to ensurethat antibodies which cross react with endogenous antibody are removed.

By “fully xenogenous antibody” is meant an antibody that has an aminoacid sequence that consists entirely of sequence xenogenous to the hostorganism (e.g., human sequence). If desired, the amount of fullyxenogenous antibody in a sample from a transgenic ungulate may bemeasured by determining the amount of antibody in the sample that (i)reacts with an antibody (e.g., anti-human immunoglobulin antibody)reactive with xenogenous antibody but (ii) does not react with anantibody (e.g., anti-bovine immunoglobulin antibody) reactive withendogenous antibody.

By “partially endogenous antibody” or “partially xenogenous antibody” ismeant an antibody that has a segment (e.g., a region of an antibodyheavy or light chain or an entire heavy or light chain) that consists ofendogenous antibody sequence and a segment (e.g., a region of anantibody heavy or light chain or an entire heavy or light chain) thatconsists of xenogenous antibody sequence. If desired, the amount ofpartially xenogenous or partially endogenous antibody in a sample from atransgenic ungulate may be measured by determining the amount ofantibody in the sample that (i) reacts with an antibody (e.g.,anti-human immunoglobulin antibody) reactive with xenogenous antibodyand (ii) reacts with an antibody (e.g., anti-bovine immunoglobulinantibody) reactive with endogenous antibody.

By “modified antibody” is meant an antibody having an altered amino acidsequence so that fewer antibodies and/or immune responses are elicitedagainst the modified antibody when it is administered to an ungulate.For example, the constant region of the antibody may be replaced withthe constant region from a bovine antibody. For the use of the antibodyin a mammal other than a bovine, an antibody may be converted to thatspecies format.

By “bifunctional antibody” is meant an antibody that includes anantibody or a fragment of an antibody covalently linked to a differentantibody or a different fragment of an antibody. In one preferredembodiment, both antibodies or fragments bind to different epitopesexpressed on the same antigen. Other preferred bifunctional antibodiesbind to two different antigens, such as to both an antibody light chainand an antibody heavy chain. Standard molecular biology techniques suchas those described herein may be used to operably link two nucleic acidsso that the fusion nucleic acid encodes a bifunctional antibody.

By “fragment” is meant a polypeptide having a region of consecutiveamino acids that is identical to the corresponding region of an antibodyof the invention but is less than the full-length sequence. The fragmenthas the ability to bind the same antigen as the corresponding antibodybased on standard assays, such as those described herein. Preferably,the binding of the fragment to the antigen is at least 20, 40, 60, 80,or 90% of that of the corresponding antibody.

By “purified” is meant separated from other components that naturallyaccompany it. Typically, a factor is substantially pure when it is atleast 50%, by weight, free from proteins, antibodies, andnaturally-occurring organic molecules with which it is naturallyassociated. Preferably, the factor is at least 75%, more preferably, atleast 90%, and most preferably, at least 99%, by weight, pure. Asubstantially pure factor may be obtained by chemical synthesis,separation of the factor from natural sources, or production of thefactor in a recombinant host cell that does not naturally produce thefactor. Proteins, vesicles, and organelles may be purified by oneskilled in the art using standard techniques such as those described byAusubel et al. (Current Protocols in Molecular Biology, John Wiley &Sons, New York, 2000). The factor is preferably at least 2, 5, or 10times as pure as the starting material, as measured using polyacrylamidegel electrophoresis, column chromatography, optical density, HPLCanalysis, or western analysis (Ausubel et al., supra). Preferred methodsof purification include immunoprecipitation, column chromatography suchas immunoaffinity chromatography, magnetic bead immunoaffinitypurification, and panning with a plate-bound antibody.

By “specifically binding a protein” is meant binding to the protein(e.g., an endogenous ungulate antibody), but not substantially bindingto other molecules (e.g., endogenous proteins other than antibodies orxenogenous antibodies) in a sample, e.g., a biological sample, thatnaturally includes the protein. Preferably, the amount antibody bound toan endogenous antibody is at least 50%, 100%, 200%, 500%, or 1,000%greater than the amount of antibody bound to an exogenous antibody underthe same conditions.

By “specifically binding mu heavy chain” is meant binding substantiallymore mu heavy chain than any other molecule in a sample. Preferably, theamount of antibody bound to an endogenous mu heavy chain is at least50%, 100%, 200%, 500%, or 1,000% greater than the amount of antibodybound to any other immunoglobulin molecule under the same conditions. Inother preferred embodiments, the binding affinity of the antibody forIgM molecules, which contain mu heavy chain, is at least 2, 5, 10, 20,or 30 fold greater than the binding affinity for IgG molecules, whichdoe not contain mu heavy chain.

By “specifically binding lambda chain” is meant binding substantiallymore lambda light chain than any other molecule in a sample. Preferably,the amount of antibody bound to an endogenous lambda light chain is atleast 50%, 100%, 200%, 500%, or 1,000% greater than the amount ofantibody bound to any other immunoglobulin molecule under the sameconditions. In other preferred embodiments, the antibody binds both IgGand IgM molecules, which both contain lambda light chain.

Advantages The present invention provides a number of advantages relatedto the production of xenogenous antibodies in transgenic ungulates. Forexample, the methods described herein have been used to express humanantibody protein in transgenic bovines. Polyclonal human antibodies thatare reactive with a specific antigen that was administered to thetransgenic bovines have also been produced. Given these promisingresults, a skilled artisan would appreciate that the methods describedherein can be used to generate other transgenic ungulates that expressdesired human antibodies (e.g., antibodies reactive with specificantigens or antibody mixtures for use as therapeutic substitute forIVIG).

The present methods also provide a simple technique for eliminatingundesired antibodies in transgenic ungulates that also express desiredxenogenous antibodies (e.g., human antibodies). By reducing the amountof endogenous antibody, these steps greatly simplify the purification ofxenogenous antibodies from the blood or milk of the transgenicungulates.

In ungulates, precursor cells only differentiate to form B-cells duringthe first half development. Thus, eliminating all of the endogenousB-cells that are present in the ungulate after this stage of developmentshould prevent any additional endogenous B-cells from being formedbecause precursor cells are no longer differentiating into B-cells.Therefore, a single dose of administered antibody may be sufficient toeliminate all of the endogenous B-cells in an ungulate. If all of theendogenous B-cells were not eliminated, additional doses of antibody maybe administered.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWING

The application file contains drawings executed in color (FIGS. 31, 34A,34C, 36A, 36B, and 42-45). Copies of this patent or patent applicationwith color drawings will be provided by the Office upon request andpayment of the necessary fee.

FIG. 1A contains an overview of the procedures used to produce a cowthat contains an Ig knockout and human artificial chromosome. The timeline in FIG. 1A is based on an estimated 18 months to prepare the Igknockout vector and generate knockout cells, 2 months to generatefetuses from the knockout cells, 9 months to perform subsequentknockouts, 9 months of gestation for calves to be born, 12 months beforeembryos can be produced from calves, and 6 months to perform the HACtransfers.

FIG. 1B contains an overview of the methods used to produce a cow thatcontains a mutation in an endogenous Ig gene and contains ΔHAC or ΔΔHAC.For the time line in FIG. 1B, it is estimated that 250 colonies arescreened per week for a total of 3,000 colonies in 3 months to isolatemale and female knockout cells. It is assumed that one or more knockoutcolonies are produced per 1,500 colonies. Homozygous knockout ungulatesmay be produced by (1) introducing a second Ig mutation in an isolatedknockout cell before nuclear transfer, (2) introducing a second Igmutation in a cell obtained from a embryo, fetus (e.g., fetus at ˜60gestation days), or offspring produced from a first round of nucleartransfer and using the resulting homozygous cell as the donor cell in asecond round of nuclear transfer, or (3) mating hemizygous ungulates. InFIGS. 1A and 1B, “Homo” denotes homozygous; “Hemi” denotes hemizygous;“H” denotes heavy chain; “L” denotes light chain; “HAC” denotes humanartificial chromosome; “HAC 1” denotes either HAC; and “HAC2” denotes asecond HAC.

FIG. 2A contains a mu (IgM heavy chain) knockout construct according tothe invention. FIG. 2B is a restriction map of immunoglobulin loci froma Holstein cattle.

FIGS. 3A and 3B contain schematic illustrations of construct “pSTneoB”and “pLoxP-STneoB” that were used to produce the mu knockout DNAconstruct, which is illustrated in FIG. 3C. FIG. 3D is thepolynucleotide sequence of the 1.5 kb region of the genomic bovine muheavy chain locus that was used as the first region of homology in themu knockout construct (SEQ ID NO: 47). FIG. 3E is the polynucleotidesequence of the 3.1 kb region of the genomic bovine mu heavy chain locusthat was used as the second region of homology in the mu knockoutconstruct (SEQ ID NO: 48). In this sequence, each “n” represents anynucleotide or no nucleotide. The region of consecutive “n” nucleotidesrepresents an approximately 0.9 to 1.0 kb region for which thepolynucleotide sequence has not been determined. FIG. 3F is a schematicillustration of a puromycin resistant, bovine mu heavy chain knockoutconstruct. FIG. 3G is the polynucleotide sequence of a bovine kappalight chain cDNA (SEQ ID NO: 60). All or part of this sequence may beused in a kappa light chain knockout construct. Additionally, this kappalight chain may be used to isolate a genomic kappa light chain sequencefor use in a kappa light chain knockout construct.

FIG. 4 is a schematic illustration of the construction of ΔHAC andΔΔHAC.

FIG. 5 is a picture of an agarose gel showing the presence of genomicDNA encoding human heavy and light chains in ΔHAC fetuses.

FIG. 6 is a picture of an agarose gel showing the expression of humanCmu exons 3 and 4 in a ΔHAC fetus at 77 gestational days (fetus #5996).

FIG. 7 is a picture of an agarose gel showing the rearrangement ofendogenous bovine heavy chain in ΔHAC fetus #5996

FIG. 8 is a picture of an agarose gel showing the expression ofrearranged human heavy chain in ΔHAC fetus #5996.

FIG. 9 is a picture of an agarose gel showing the expression of thespliced constant region from the human heavy chain locus in ΔHAC fetus#5996

FIG. 10 is a picture of an agarose gel showing the expression ofrearranged human heavy chain in ΔHAC fetus #5996.

FIG. 11A is the polynucleotide sequence of a rearranged human heavychain transcript from ΔHAC fetus #5996 (SEQ ID NO: 49). FIG. 11B is asequence alignment of a region of this sequence (“Query”) with a humananti-pneumococcal antibody (“Sbjct”) (SEQ ID NOs: 50 and 51,respectively). For the query sequence from ΔHAC fetus #5996, only thosenucleotides that differ from the corresponding nucleotides of the humananti-pneumococcal antibody sequence are shown.

FIGS. 12A and 12B are two additional polynucleotide sequences (SEQ IDNOs: 52 and 54) and their deduced amino acid sequences (SEQ ID NOs: 53and 55, respectively) of rearranged human heavy chain transcripts fromΔHAC fetus #5996.

FIG. 13 is a picture of an agarose gel demonstrating that ΔΔHAC fetus#5580 contains both human heavy and light chain immunoglobulin loci.

FIG. 14 is a picture of an agarose gel demonstrating that ΔΔHAC fetuses#5442A, and 5442B contain both human heavy and light chain loci.

FIG. 15 is a picture of an agarose gel showing the expression of thespliced mu constant region from the human heavy chain locus in ΔΔHACfetus #5542A.

FIG. 16 is a picture of an agarose gel showing the rearrangement andexpression of the human heavy chain locus in ΔΔHAC fetus #5868A.

FIG. 17 is a picture of an agarose gel showing rearrangement andexpression of the human Ig lambda locus in ΔΔHAC fetuses #5442A and5442B.

FIG. 18 is a picture of an agarose gel showing rearrangement andexpression of the human Ig lambda locus in ΔΔHAC fetus #5442A.

FIG. 19 is a picture of an agarose gel showing rearrangement andexpression of the human Ig lambda locus in ΔΔHAC fetus #5868A.

FIG. 20 is a polynucleotide sequence and the corresponded deduced aminoacid sequence of a rearranged human light chain transcript from ΔΔHACfetus #5442A (SEQ ID NOs: 56 and 57, respectively).

FIG. 21 is another polynucleotide sequence and the corresponding deducedamino acid sequence of a rearranged human light chain transcript fromΔΔHAC fetus #5442A (SEQ ID NOs: 58 and 59, respectively).

FIGS. 22A-22H are graphs of a FACS analysis of expression of humanlambda light chain and bovine heavy chain proteins by ΔΔHAC fetuses#5442A (FIGS. 22A-22D) and 5442B (FIGS. 22E-22H). Lymphocytes from thespleens of these fetuses were reacted with a phycoerytherin labeledanti-human lambda antibody (FIGS. 22C and 22D), a FITC labeledanti-bovine IgM antibody (FIGS. 22D and 22H), or no antibody (FIGS. 22A,22B, (22E, and 22F) and then analyzed on a FASCalibur cell sorter. Thepercent of cells that were labeled with one of the antibodies isdisplayed beneath each histogram.

FIG. 23 is a schematic illustration of the α-(1,3)-galactosyltransferaseknockout vector used to insert a puromycin resistance gene and atranscription termination sequence into the endogenousα-(1,3)-galactosyltransferase gene in bovine cells.

FIG. 24 is a schematic illustration of a BamHI-XhoI fragment containingexons 2, 3, and 4 that was used as a backbone for the AAV targetingvector. A neomycin resistance marker was used for insertionalmutagenesis of the locus by insertion into exon 4. The location of theannealing sites for the PCR primers that were used for subsequentconfirmation of appropriate targeting is indicated.

FIG. 25 is a schematic illustration of the construction of anadeno-associated viral construct designed to remove endogenous bovineIgH sequence.

FIG. 26 is a picture of an agarose gel showing the PCR analysis ofindividually transduced clones for appropriate targeting events. Thevector used in this experiment is shown in FIG. 24. PCR productsindicative of appropriate targeting are marked with asterisks.

FIG. 27 is a table listing pregnancy rates for HAC carrying embryos.

FIGS. 28A-28J are pictures of FACS analysis of peripheral bloodlymphocytes from either experimental fetuses injected with ananti-bovine IgM antibody (das6) to inhibit B-cell development or controlfetuses. FIGS. 28A-28E are pictures of the FACS analysis performed usingan anti-bovine IgM antibody to detect IgM molecules expressed on thesurface of B-cells. As illustrated in FIG. 28A and FIG. 2B,approximately 19.82 to 26.61% of the peripheral blood lymphocytes fromthe control fetuses expressed IgM. In contrast, 7.78, 11.80, or 3.95% ofthe peripheral blood lymphocytes from the three fetuses injected withthe anti-bovine IgM antibody expressed IgM (FIGS. 28C-28E,respectively). FIGS. 28F-28J are pictures of the FACS analysis performedusing an anti-bovine light chain antibody (das10) to detect antibodylight chain molecules expressed on the surface of B-cells. Asillustrated in FIG. 28F and FIG. 28G, approximately 12.43 to 29.47% ofthe peripheral blood lymphocytes from the control fetuses expressedantibody light chain molecules. In contrast, 2.54, 13.77, or 3.99% ofthe peripheral blood lymphocytes from the three fetuses injected withthe anti-bovine IgM antibody expressed antibody light chain molecules(FIGS. 28H-28J, respectively).

FIGS. 29A and 29B illustrate the immunodetection of nuclear envelope andnuclear matrix proteins in bovine preimplantation embryos. FIG. 29A is apicture of in vitro-fertilized bovine embryos at the pronuclear and8-cell stage examined using the same antibodies. Arrows in FIG. 29A toanti-NuMA and anti-AKAP95 labeling in the female pronucleus ofpronuclear stage embryos. Insets in FIG. 29A are pictures of DNA labeledwith 0.1 μg/ml Hoechst 33342 (bars, 20 μm). FIG. 29B is theimmunoblotting analysis of bovine fibroblasts (upper rows) andpronuclear stage embryos (lower rows). Molecular weight markers areshown in kDa on the right of FIG. 29B.

FIG. 30 illustrates the dynamics of the nuclear envelope, NuMA, andAKAP95 during premature chromatin condensation and pronuclear assemblyin nuclear transplant embryos. FIG. 30 is a picture of bovine donorfibroblasts (Donor cell), nuclear transplant embryos at the prematurechromatin condensation stage (three hours post-fusion), nucleartransplant embryos at the pronuclear stage (19 hours post-fusion), andparthenogenetic pronuclear stage embryos activated as described herein.Disassembly of the donor nucleus and assembly of the new pronuclei weremonitored at the premature chromatin condensation stage three hours postinjection “hpi” (“PCC”) and seven hours post injection (“NT PN”), usinganti-lamin B, lamins A/C, NuMA, and AKAP95 antibodies. Female pronucleiformed after parthenogenetic activation of MII oocytes with 10 mM SrCl₂were also analyzed five hours after start of activation treatment(“Parth. PN”). Lamins A/C were assembled in pronuclei of bovinepronuclear stage nuclear transplant embryos. DNA was counterstained with0.1 μg/ml Hoechst 33342. TRITC refers to labeling with TRITC-conjugatedsecondary antibodies (bars, 20 μm).

FIG. 31 is a graph demonstrating that AKAP95 is more strongly anchoredin pronuclei of nuclear transplant embryos compared to parthenogeneticembryos. This graph shows the relative percent of unextracted lamin B,AKAP95, and DNA labeling in pronuclei of parthenotes, nuclear transplantembryos, and somatic donor nuclei after in situ extraction with 0.1%Triton X-100 and 1 mg/ml DNAse I together with 100 or 300 mM NaCl for 30minutes at room temperature prior to fixation with 3% paraformaldehyde.Localization of B-type lamins (red) and AKAP95 (green) was examined bydouble immunofluorescence. Fluorescence labeling intensity in eachchannel—red, (lamin B), blue (DNA), and green (AKAP95)—was quantified.The reference value (100% unextracted) represents relative amounts ofB-type lamins, DNA, and AKAP95 staining in embryos or cellspermeabilized with 0.1% Triton X-100 only prior to fixation.Approximately 30 embryos were examined in each group.

FIG. 32 demonstrates that lamins A/C are transcribed de novo uponpronuclear reconstitution in nuclear transplant embryos. FIG. 32 is apicture of bovine pronuclear nuclear transplant embryos produced byfibroblast fusion and oocyte activation with either 5 μM ionomycin forfour minutes followed by 10 μg/ml cycloheximide/2.5 μg/ml cytochalasin Dfor four hours (b′, -), ionomycin/cycloheximide/cytochalasin D as in(b′) followed by an additional nine hours of culture with 10 μg/mlcycloheximide (b″, CHX) or incubation as in (b′) together with 1 μg/mlactinomycin D during the entire activation treatment (b′″). Anti-lamin B(rabbit polyclonal) and anti-lamins A/C (mAb) antibodies were used onthe same preparations. Insets are pictures of DNA labeling with 0.1μg/ml Hoechst 33342 (bars, 20 μm).

FIG. 33 is a graph of chromosome condensation and nuclear envelopebreakdown in mitotic cytoplasmic extract (M-S15), mitotic cytosolicextract (M-S200), and oocyte extract (MII-S15) (n=300-400 nucleiexamined in 3-5 replicates).

FIGS. 34A-34C are sets of pictures of immunofluorescence analysis ofpurified input bovine fibroblast nuclei (FIG. 34A) and condensedchromatin produced in mitotic cytosolic extract (FIG. 634) and oocyteextract (FIG. 34C). The indicated nuclear markers were examined. DNA wascounterstained with propidium iodide (red) (bars, 10 μm).

FIG. 35 is a set of pictures of immunofluorescence analysis of condensedchromatin obtained in oocytes following conventional nuclear transplant(NT) or nuclear injection (NI) methods and following injection ofchromatin masses into oocytes (CT) using the methods of the presentinvention. Both detectable lamins B and A/C appear to be solubilized(bar, 10 μm).

FIGS. 36A and 36B are sets of pictures of immunofluorescence analysis ofpronuclei resulting from chromatin transfer, nuclear transplant, ornuclear injection. Embryos were fixed at 19 hours post nucleartransplant, nuclear injection, or chromatin transfer and labeled.Control parthenogenetic pronuclei (Part.) were also examined.

FIG. 36A shows the analysis of lamins A/C and B. FIG. 36B shows theanalysis of AKAP95 and NuMA. Lamins A/C (green label) only appear innuclear transplant and nuclear injection pronuclei (bars, 30 μm).

FIG. 37 is a diagram of a procedure for production of cloned Tc calves.Construction of HAC is shown with hChr22 and hChr14 regions containingIgλ and IgH genes and the position of the neo selection marker. From aCHO clone, the HAC was transferred into fetal bovine fibroblasts bymeans of a MMCT technique. Tc fibroblasts were fused with an enucleatedoocyte for nuclear transfer. The reconstituted Tc embryos were culturedin vitro to the blastocyst stage then implanted into recipient cows.Around 60 days of gestation Tc fetuses were recovered; fibroblast celllines were reestablished, evaluated and used for further nucleartransfer. Recloned Tc embryos were transferred to recipients to produceTc calves.

FIGS. 38A-38D illustrate the analysis of Tc fetuses. G418 selection ofregenerated Tc fibroblast line and control non-transgenic fibroblasts(FIG. 38A). Genomic PCR of IgH and Igl loci in Tc fetuses and controls.The three fetuses, #5968 (lane 1), #6032 (lane 2) and #6045 (lane 3)were derived from ΔHAC fibroblasts, fetus #5580 (lane 4) was from ΔΔHACfibroblasts. As a control, a non-transgenic fetus (lane N) was recoveredand evaluated (FIG. 38B). In all Tc fetuses and a positive control humanliver DNA sample (lane P), both human IgH and Igl loci were detected byPCR, but not in the negative control (lane N). Rearranged and expressedhuman Igμ and Igλ transcripts amplified by RT-PCR from negative controlnon-transgenic bovine spleen (lane N), from brain (lane 1), liver (lane2) and spleen (lane 3) of cloned Tc fetus and positive control humanspleen (lane P) (FIG. 38C). A representative nucleotide and deducedamino acid sequence of human Igμ and Igλ transcripts amplified by RT-PCRfrom a cloned Tc fetus recovered at 91 days (FIG. 38D). RT-PCR wascarried out as described (Kuroiwa et al., Nature Biotech 18:1086-1090,2000). For human Igμ transcripts, VH1/5 BACK, VH3 BACK, and VH4BACK wereused as a 5′ primers and Cμ-2 was used as a 3′ primer. For human Igλtranscripts, Vλ1LEA1, Vλ2MIX and Vλ3MIX were used as 5′ primers, andCλMIX was used as a 3′ primer. The amplified cDNAs were subcloned usinga TA cloning kit (Invitrogen) and sequenced using a DNA autosequencer(ABI3700 system).

FIGS. 39A-39C are pictures illustrating the analysis of cloned Tccalves. Four cloned Tc calves; male calf (#50) from cell line #6045 andfemale calves (#1064, #1065, #1066) from cell line #5968 (FIG. 39A).Genomic PCR of IgH and Igλ loci from PBLs from cloned Tc calves andcontrols; calf #1064 (lane 1), #1065 (lane 2), #1066 (lane 3), #50 (lane4), #1067 (lane 5) and #1068 (lane 6) (FIG. 39B). In all the Tc calvesand positive control human liver DNA (lane P) both human IgH and Igλloci were detected by genomic PCR, but not in a negative control,non-transgenic calf (lane N). FISH analysis in metaphase chromosomespreads in a cell showing a single signal and a cell showing a doublesignal (FIG. 39C). Arrows indicate location of HACs amongst surroundingbovine chromosomes. HAC painting was done using digoxigenin labeledhuman COT-1 DNA as a probe and detected with ananti-digoxigenin-rhodamine.

FIGS. 40A and 40B are schematic diagrams of an IgM knockout vector witha puromycin-resistance gene and a strategy for identifying correctlytargeted cells using this vector, respectively. FIGS. 40C and 40D areschematic diagrams of an IgM knockout vector with a neomycin-resistancegene and a strategy for identifying correctly targeted cells using thisvector, respectively.

FIG. 41 is a bar graph illustrating the effect of immunizing a ΔHACtransgenic animal with DNP-KLH. In particular, adjuvant-stimulatedimmunization of HAC-transchromosomal cattle generates a polyclonal,antigen-specific response to the immunizing antigen. The reaction of theantibody to different epitopes of the immunizing antigen demonstratesthat HAC transchromosomal animals can recognize multiple epitopes of acomplex antigen. A ΔHAC 1066 bovine was immunized subcutaneously with400 ug of an exemplary antigen, DNP-KLH, 2,4 dinitrophenylated keyholelimpet hemocyanin, and Complete Freund's Adjuvant. The animal was thenimmunized a second time with 400 ug of DNP-KLH, without any adjuvant.Serum was collected from the immunized animal and analyzed forreactivity with both hapten and carrier components of the exemplaryantigen, DNP-BSA and KLH, respectively. Such a pattern of reactivity ischaracteristic of a polyclonal antibody that is made up of a mixture ofantibodies that recognize multiple and different epitopes of a complexantigen. Human immunoglobulin was affinity purified from collected serausing a bovine-anti-human Ig column. Equimolar concentrations ofaffinity purified human Ig from ΔHAC 1066 was tested using solid phaseELISA for reactivity with DNP-BSA, KLH, or BSA (negative control). Aspecies specific bovine anti-human biotinylated polyclonal antibody wasused as the detection reagent. Equimolar concentrations of commerciallyproduced human Ig and bovine Ig were analyzed in parallel as negativecontrols. Optical densities at 405 nm were taken and graphed for allsamples. Only human Ig from a ΔHAC animal immunized with DNP-KLH (1066)demonstrated specific reactivity to antigen. Commercially producedbovine Ig and human Ig did not react with DNP-KLH and resembledbackground levels. A PBS-BSA plate was also used as a negative controland showed no reactivity.

FIG. 42 is a picture of a gel illustrating the purification of humanantibodies from HAC serum. In particular, fully human antibodies can bepurified from the serum of HAC transchromosomal cattle. Furthermore, thepresence of mu and gamma heavy chains demonstrates that the HACundergoes class switching and that the cloned transchromosomic hostsupports class switching at the human heavy chains locus.

Human antibody from ΔHAC 82 was affinity purified using a bovineanti-human Ig column and applied to a denaturing protein gel for westernanalysis. Duplicate blots with equal amounts (1 ug), of all samples wereanalyzed in parallel. Western blot analysis was performed using twodetection reagents in independent duplicate blots. The two polyclonalspecies specific reagents used were (i) biotinylated bovine anti-humanIg and (ii) biotinylated horse anti-bovine Ig. The following controlswere applied to each gel: human IgG, IgM, and a mixture of bovine Igfrom commercial sources. When probed with bovine anti-human Ig, thepurified human Ig from a ΔHAC animal produced bands which migrated atmolecular weights that matches those of both human heavy chains, μ and γas well as human light chain. This bovine anti-human detecting reagentreacts specifically with human Ig without cross-reacting with bovine Ig.In the preparation of human Ig from the ΔHAC animal, bovine Ig was notdetected using the horse anti-bovine reagent (detection limits of thisreagent are greater than or equal to 50 ng bovine Ig). This horseanti-bovine detecting reagent reacts specifically with bovine Ig withoutcross-reacting with human Ig. The observation of human IgM and IgG inthe HAC human Ig preparation demonstrates Ig class-switchingcapabilities. In 1 ug of HAC human Ig, no significant levels of bovineIg determinants were observed above the detection limit of this reagent.

FIG. 43 illustrates that the HAC-encoded human light chains (lambda) arebound to human mu chains and human gamma chains. This resultdemonstrates that the xenogeneic B cells of HAC-transchromosomal animalsare capable of assembling human heavy and light chains. In particular,purified human Ig from ΔHAC 82 show both human γ and μ liked directly tohuman λ chains. Human antibody from ΔHAC 82 was affinity purified usinga bovine anti-human Ig column. Equimolar concentrations (100 ng/ml) ofpurified HAC human Ig were tested in two solid-phase ELISAs. The capturereagent for Plate A was a purified monoclonal mouse anti-human μ Ig, andthe capture reagent for Plate B was a mouse anti-human γ Ig, both coatedat a concentration of 10 ug/ml. As controls for each assay, equimolarconcentrations of commercially produced purified bovine Ig and human Ig(100 ng/ml) were analyzed in parallel. The detecting reagent for bothassays was a biotinylated monoclonal mouse anti-human λ. In each assay,human Ig from ΔHAC 82 exhibited levels above background (e.g., levelsabove bovine IgG/M and BSA Plate C). Both monoclonal reagents arespecific for their recognized human heavy chain class and do notcross-react with bovine Ig.

FIG. 44 illustrates that human mu, gamma, and light chains are found inhuman Ig purified from a HAC-transchromosomal calf in which anantigen-specific human antibody response has been induced. Inparticular, purified human antibodies from DNP-KLH immunized HAC serumwas characterized. Human antibody from ΔHAC 1066 was affinity purifiedusing a bovine anti-human Ig column and then analyzed via western forhuman Ig. Approximately 1 ug (1×) and 5 ug (5×) of human Ig from theDNP-KLH immunized animal resolved on a PAGE gel. The commerciallyproduced human IgG and IgM (1 ug each) were analyzed in parallelanalysis. The polyclonal species-specific reagent, biotinylated bovineanti-human Ig, was used to detect human Ig. Human Ig from the ΔHAC 1066animal yield bands which migrate at a molecular weight that correspondsto those of both human heavy chains μ and γ as well as human lightchain. The presence of μ and γ demonstrates the capacity of theHAC-resident human heavy chain locus undergo class switching and theability of HAC transchromosomal cattle to effect class switching of thishuman Ig locus. This detection reagent reacts specifically with human Igwithout cross-reacting with bovine Ig.

FIG. 45 illustrates the SLOT procedure. In step one, donor fibroblastsare reversibly permeabilized for 30 minutes with 500 ng/ml SLO. In steptwo, permeabilized cells are washed and incubated in a mitotic extractcontaining an ATP-regenerating system to elicit chromosome condensationand promote removal of nuclear components (arrows). In step three, theextract is removed, and the cells are optionally resealed in culturewith 2 mM CaCl₂ for two hours. In step four, cells are fused toenucleated recipient oocytes, and in step five, oocytes are activated asfor NT to elicit pronuclear formation and development.

DETAILED DESCRIPTION

Methods for producing ungulates that express xenogenous antibodiesVarious approaches may be used to produce ungulates that expressxenogenous (e.g., human) antibodies. These approaches include, forexample, the insertion of a human artificial chromosome (HAC) containingboth heavy and light chain immunoglobulin genes into an ungulate or theinsertion of human B-cells or B-cell precursors into an ungulate duringits fetal stage or after it is born (e.g., an immune deficient or immunesuppressed ungulate) (see, for example, WO 01/35735, filed Nov. 17,2000, US 02/08645, filed Mar. 20, 2002). In either case, both humanantibody producing B-cells and ungulate antibody-producing B-cells maybe present in the ungulate. In an ungulate containing a HAC, a singleB-cell may produce an antibody that contains a combination of ungulateand human heavy and light chain proteins.

Standard methods can be used to introduce a desired nucleic acid whichcontains genes (preferably, entire gene loci) for producing antibodiesof a particular species (e.g., a human) into a donor cell for use ingenerating a transgenic ungulate that expresses both xenogenous andendogenous antibodies. Preferably, human artificial chromosomes areused, such as those disclosed in WO 97/07671 (EP 0843961) and WO00/10383(EP 1106061). These human artificial chromosomes also are described in acorresponding issued Japanese patent JP 30300092. Both of theseapplications are incorporated by reference in their entirety herein.Also, the construction of artificial human chromosomes that contain andexpress human immunoglobulin genes is disclosed in Shen et al., Hum.Mol. Genet. 6(8):1375-1382 (1997); Kuroiwa et al., Nature Biotechnol.18(10):1086-1090 (2000); Loupert et al., Chromosome 107(4):255-259(1998); WO00/10383 (EP 1106061); WO98/24893; WO96/33735; WO 97/13852;WO98/24884; WO97/07671 (EP 0843961); U.S. Pat. No. 5,877,397; U.S. Pat.No. 5,874,299; U.S. Pat. No. 5,814,318; U.S. Pat. No. 5,789,650; U.S.Pat. No. 5,770,429; U.S. Pat. No. 5,661,016; U.S. Pat. No. 5,633,425;U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,569,825; and U.S. Pat. No.5,545,806; all of which are incorporated by reference in their entiretyherein. Human artificial chromosomes may also be utilized to introducexenogenous antibody genes into wild-type animal cells; this isaccomplished using the methods described above. Introduction ofartificial chromosome into animal cells, especially fetal fibroblastcells can be performed by microcell fusion as described herein.

In an alternative to the use of human artificial chromosome, nucleicacid encoding immunoglobulin genes may be integrated into the chromosomeusing a YAC vector, BAC vector, or cosmid vector. Vectors comprisingxenogenous Ig genes (WO98/24893, WO96/33735, WO 97/13852, WO98/24884;U.S. Pat. No. 5,849,992 issued Dec. 15, 1998 to Meade et al., and U.S.Pat. No. 5,827,690 issued Oct. 27, 1998 to Meade et al.) can beintroduced to fetal fibroblasts cells using known methods, such aselectroporation, lipofection, fusion with a yeast spheroplast comprisinga YAC vector, and the like. Further, vectors comprising xenogenous Iggenes can be targeted to the endogenous Ig gene loci of the fetalfibroblast cells, resulting in the simultaneous introduction of thexenogenous Ig gene and the disruption of the endogenous Ig gene.

Integration of a nucleic acid encoding a xenogenous immunoglobulin genemay also be carried out as described in the patents by Lonberg et al.(supra). In the “knockin” construct used for the insertion of xenogenousimmunoglobulin genes into a chromosome of a host ungulate, one or moreimmunoglobulin genes and an antibiotic resistance gene may beoperably-linked to a promoter which is active in the cell typetransfected with the construct. For example, a constitutively active,inducible, or tissue specific promoter may be used to activatetranscription of the integrated antibiotic resistance gene, allowingtransfected cells to be selected based on their resulting antibioticresistance. Alternatively, a knockin construct in which the knockincassette containing the Ig gene(s) and the antibiotic resistance gene isnot operably-linked to a promoter may be used. In this case, cells inwhich the knockin cassette integrates downstream of an endogenouspromoter may be selected based on the resulting expression of theantibiotic resistance marker under the control of the endogenouspromoter. These selected cells may be used in the nuclear transferprocedures described herein to generate a transgenic ungulate containinga xenogenous immunoglobulin gene integrated into a host chromosome.

Using similar methodologies, it is possible to produce and insertartificial chromosomes containing genes for expression of Ig ofdifferent species such as dog, cat, other ungulates, non-human primatesamong other species. As discussed above, and as known in the art,immunoglobulin genes of different species are well known to exhibitsubstantial sequence homology across different species.

Once it has been determined that the inserted artificial chromosome, forexample, a human artificial chromosome, has been stably introduced intoa cell line, e.g., a bovine fetal fibroblast, it is utilized as a donorfor nuclear transfer. This may be determined by PCR methods. Theresulting transgenic calves comprise a stably introduced nucleic acid,such as a human artificial chromosome. After calves have been obtainedwhich include the stably incorporated nucleic acid (for example, humanartificial chromosome), the animals are tested to determine whether theyexpress human Ig genes in response to immunization and affinitymaturation.

Modifications of the overall procedure described above may also beperformed. For example, to reduce the amount of endogenous antibodyexpressed by the resulting transgenic ungulate, one or more endogenousIg genes may be inactivated in the donor cell before or after insertionof the xenogenous nucleic aicd. Further, an animal retaining xenogenousIg genes may be mated with an animal in which an endogenous Ig gene isinactivated.

Alternative methods for generating an ungulate that expresses bothendogenous and xenogenous antibodies involve remodeling the donorgenetic material before it is inserted into a recipient oocyte to formthe transgenic ungulate. Remodeling refers to any morphological changethat improves development of the resulting nuclear transplant oocyteover that derived from either transferring whole cells or intact nucleiinto a recipient oocyte. Reprogramming is achieved by incubating a donornucleus that contains a xenogenous immunoglobulin nucleic acid in areprogramming media (e.g., a mitotic extract, detergent and/or saltsolution, or protein kinase solution) resulting in nuclear envelopedissolution and possibly chromatin condensation. This nuclear envelopebreakdown and chromatin condensation allows the release of transcriptionregulatory proteins that were attached to the chromosomes and that wouldotherwise promote the transcription of genes undesirable for oocyte,embryo, or fetus development. Additional regulatory proteins may beremoved by purifying the chromatin mass prior to transferring it into arecipient oocyte. Alternatively, specific regulatory proteins that arereleased from the chromosomes may be immunodepleted or otherwise removedfrom the reprogrammed media (e.g., a cell extract) to prevent them fromre-binding the chromosomes. After nuclear transfer, new proteins fromthe oocyte cytoplasm may be bound to the chromosomes duringdecondensation of the chromatin and nuclear envelope formation in theoocyte. These proteins promote the transcription of genes that allow theoocyte to develop into a viable offspring.

Another cloning method that can be used to produce the transgenicungulate involves reprogramming a permeabilized cell (i.e., a cellcontaining a xenogenous immunoglobulin nucleic acid) by incubating it ina reprogramming media (e.g., a cell extract) to allow the addition orremoval of factors from the cell. The plasma membrane of thepermeabilized cell is preferably resealed to enclose the desired factorsand restore the membrane integrity of the cell. The reprogrammed cell isthen transferred into a recipient oocyte for the production of a clonedmammal. This cloning method has been used to produce fetuses without axenogenous immunoglobulin nucleic acid that have survived past day 60.Preliminary results indicate that fetal survival between day 40 and day60 is higher for fetuses formed using this method (7/10; 70%) than forconventional nuclear transfer fetuses (8/16; 50%). Similar results areexpected for fetuses with a xenogenous immunoglobulin nucleic acid.

The methods of the invention can also be used to reduce the amount ofendogenous antibodies produced by ungulates derived from chimericembryos that have genes for both xenogenous and endogenous antibodies.Chimeric embryos in which the majority of the placental tissue is fromone genetic source and the majority of the fetal tissue is from anothergenetic source can be generated. These chimeric embryos may have fewerplacental abnormalities and thus may have an increased survival rate. Inone such method, cells from an in vitro fertilized ornaturally-occurring embryo are contacted with cells from an embryo thathas a nucleic acid encoding a xenogenous antibody and that is producedusing traditional nuclear transfer methods or any of the other cloningmethods described herein. For example, cells from an in vitro fertilizedembryo can be injected into the periphery of a nuclear transfer embryo(e.g., between the zona pellucida and the embryo itself). This methodwas used to produce chimeric embryos without xenogenous antibody genesthat had a 67% survival rate at day 40 compared to a 25% survival ratefor control nuclear transfer embryos. Similar results are expected usingcells from nuclear transfer embryos that have a xenogenous antibodygene. In an alternative method, cells from a precompaction, in vitrofertilized or naturally-occurring embryo are incubated with cells from aprecompaction nuclear transfer embryo under conditions that allow cellsfrom each embryo to reorganize to produce a single chimeric embryo(Wells and Powell, Cloning 2:9-22, 2000). In both methods, the cellsfrom the in vitro fertilized or naturally-occurring embryo arepreferentially incorporated into the placenta, and the cells from thenuclear transfer method are preferentially incorporated into the fetaltissue.

Sequential manipulation of a donor cell (e.g., a bovine fetal fibroblastcell) is useful for generating a human antibody-producing bovine, withor without mating offspring. Sequential manipulation of a donor cellincludes the process of (i) manipulation of a donor cell, (ii) nucleartransfer or chromatin transfer, (iii) generation of a fetus, and (iv)isolation of a donor cell from the fetus, such as a fetal fibroblast. Inone particular embodiment, a heavy chain hemizygous KO fetal fibroblastis used as a donor cell in nuclear or chromatin transfer cloningmethods. A cell from the resulting fetus is modified to generate a heavychain homozygous KO fetal fibroblast. This fibroblast is used as a donorcell in nuclear or chromatin transfer methods, and a fibroblast from theresulting fetus is genetically modified to generate a light chainhemizygous KO fetal fibroblast. After nuclear or chromatin transfer, afibroblast is genetically modified to produce a light chain homozygousKO fetal fibroblast, which is then used as a donor cell in nuclear orchromatin transfer. A HAC is introduced into a fibroblast from theresulting fetus, and the HAC-containing fibroblast is used in nuclear orchromatin transfer to generate a calf that produces human antibodies.

With respect to the practical production of human antibodies,inactivation of bovine light chain nucleic acids is typically notrequired because the desired fully human antibodies can be separatedfrom undesired antibodies with bovine light chains using a bovineanti-human IgL affinity column. Also, our recent data suggests that theamount of chimeric molecules (e.g., antibodies with both bovine andhuman sequences) may be much less than that of fully human antibodies.If inactivation of bovine light chain nucleic acids is desired, bovinelambda light chain can be mutated using the methods described herein.

Methods for producing xenogenous antibodies in ungulates and eliminatingundesired endogenous antibodies As discussed above, the presentinvention also relates to the production of a transgenic ungulate,preferably a transgenic cow, wherein (i) endogenous Ig expression hasbeen reduced by administering an antibody that inhibits endogenousB-cells or antibodies and (ii) a nucleic acid (e.g., an artificialchromosome) has been stably introduced that comprises genes which arenecessary for the production of functional antibodies of anotherspecies, (e.g., human). Thereby, a transgenic animal may be obtainedthat does not produce its endogenous antibodies, but which insteadproduces antibodies of another species. Any non-endogenous antibodiesmay be produced including, without limitation, human, non-human primate,dog, cat, mouse, rat, or guinea pig antibodies.

In particular, a compound that reduces the production of endogenousantibodies by B-cells or that reduces the number of functional,endogenous B-cells may be administered to a non-human mammal (e.g., anungulate). This immunodepletion can be performed by injecting theungulate with either monoclonal or polyclonal antibodies againstendogenous IgM heavy and/or endogenous light chains (e.g., lambda and/orkappa light chains). B-cells expressing either endogenous heavy or lightchain proteins on their surface are susceptible to either humoral orcell-mediated elimination, apoptosis, or antibody-mediated cytotoxinuptake. The population of fully xenogenous (e.g., human) antibodyexpressing B-cells may then expand to maintain antibody levels. Forexample, we demonstrated that the injection of an anti-bovine IgMantibody into fetuses reduced the number of peripheral blood B-cells.These compounds may be administered during the normal period ofdevelopment of the mammal's immune system (e.g., during the fetal,embryonic, or postnatal stage) or after this period to inhibit thedevelopment of endogenous B-cells in the ungulate.

This method is preferably applied to ungulates that express bothendogenous and xenogenous antibodies to increase the percentage ofxenogenous antibody produced by the ungulate. Reducing the amount ofungulate and ungulate/xenogenous chimeric antibody molecules producedenhances the quantity of human antibody production and simplifypurification of fully human antibody from ungulate blood or milk. Forexample, a polyclonal antibody against bovine antibodies can be injectedinto a bovine expressing some fully bovine, some fully human, and somebovine/human chimeric antibody molecules to deplete bovine andbovine/human chimeric antibody producing B-cells. The bovine would thenbe enriched for production of fully human antibodies.

State of the art prior to present invention While human Ig has beenexpressed in mice, it was unpredictable whether human Ig will befractionally rearranged and expressed in bovines, or other ungulates,because of differences in antibody gene structure, antibody productionmechanism, and B-cell function. In particular, unlike mice, cattle andsheep differ from humans in their immunophysiology (Lucier et al., J.Immunol. 161: 5438, 1998; Parng et al., J. Immunol. 157:5478, 1996; andButler, Rev. Sci. Tech. 17:43, 2000). For example antibody genediversification in bovines and ovines relies much more on geneconversion than gene rearrangement as in humans and mice. Also, theprimary location of B-cells in humans and mice is in the bone marrow,whereas in bovines and ovines B-cells are located in the illeal Peyer'spatch. Consequently, it would have been difficult, if not impossible,prior to the present invention, to predict whether immunoglobulinrearrangement and diversification of a human immunoglobulin loci wouldtake place within the bovine (or other ungulate) B-cell lineage. Inaddition, it would also have been unpredictable whether a bovine wouldbe able to survive, i.e., elicit its normal immune functions, in theabsence of its endogenous Ig or with interference from human antibodies.For example, it was not certain if bovine B-cells expressing human Igwould correctly migrate to the illeal Peyer's Patch in bovines becausethis does not happen in humans. Also, it is not clear if human Fcreceptor function; which mediates complement activation, induction ofcytokine release, and antigen removal; would be normal in a bovinesystem. It was unpredictable whether such ungulates would survivebecause it is uncertain whether human Igs will be functionallyexpressed, or expressed in sufficient amounts to provide for adequateimmune responses. Also, it was uncertain whether human chromosomes willbe stably maintained in transgenic ungulates.

Still further, it was uncertain whether ungulate (for example, bovine)B-cells will be able to express or properly rearrange human or othernon-endogenous Igs. It was also unpredictable whether allelic exclusionin transgenic ungulates would produce desired B-cells that express fullyxenogenous antibody. This desired, fully xenogenous antibody is noteliminated from the ungulate when the methods described herein are usedto administer an antibody to eliminate undesired, endogenous antibody.In contrast, if this allelic exclusion does not occur, the ungulatewould express only partially xenogenous or fully endogenous antibodies.In this latter case, an antibody administered to the ungulate toeliminate endogenous antibodies might eliminate the partially xenogenousantibodies as well as the fully endogenous antibodies.

While the approaches to be utilized in the invention have been describedabove, the techniques that are utilized are described in greater detailbelow. These examples are provided to illustrate the invention, andshould not be construed as limiting. In particular, while these examplesfocus on transgenic bovines, the methods described may be used toproduce and test any transgenic ungulate.

EXAMPLE 1 Introduction and Rearrangement of HAC Summary of Proceduresfor Insertion of HACs

For the generation of ungulates that express xenogenous antibody andthat optionally have a mutation in an endogenous antibody gene, standardmethods may be used to insert a nucleic acid encoding a xenogenousantibody (e.g., a HAC) into a cell. If desired, one or more endogenousantibody genes may be mutated in the cell. The cell is then used instandard nuclear transfer procedures to produce the desired transgenicungulate, as described in more detail below.

Essentially, male and female bovine fetal fibroblast cell linescontaining human artificial chromosome sequences (e.g., #14fg., #2fg.,and #22fg.) are obtained and selected and used to produce cloned calvesfrom these lines.

For example, HACs derived from human chromosome #14 (“#14fg,” comprisingthe Ig heavy chain gene), human chromosome #2 (“#2fg,” comprising the Igkappa chain gene) and human chromosome #22 (“#22fg,” comprising the Iglambda chain gene) can be introduced simultaneously or successively.

The transmission of these chromosome fragments is tested by mating amale #14fg. animal to female #2fg. and #22fg. animals and evaluatingoffspring. If transmission is successful then the two lines are mated toproduce a line containing all three chromosome fragments.

Also, #14fg., #2fg., and #22fg. chromosome fragments may be insertedinto fetal cells (e.g., transgenic Homo H/L fetal cells) and used togenerate cloned calves or cross transgenic HAC calves with othertransgenic calves (e.g., Homo H/L calves). Alternatively, other HACs,such as ΔHAC or ΔΔHAC, may be introduced as described below orintroduced using any other chromosome transfer method.

Rationale Germline transmission of HACs should be useful for introducingthe HACs into the animals (e.g., Ig knockout animals) and in propagatinganimals in production herds. The concern in propagation of HACs throughthe germline is incomplete pairing of chromosomal material duringmeiosis. However, germline transmission has been successful in mice asshown by Tomizuka et al. (Proc. Natl. Acad. Sci. USA, 97:722, 2000).

The strategy outlined in FIG. 1A consists of inserting #14fg. into amale line of cells and #2fg. and #22fg. each into female cell lines.Calves retaining a HAC are produced and germline transmission can betested both through females and males. Part of the resulting offspring(˜25%) should contain both heavy and light chain HACs. Further crossingshould result in a line of calves containing all three chromosomalfragments. These animals are optionally used for crossing withtransgenic animals (e.g., Homo H/L animals), produced from fetal cellsas previously described.

Experimental Design Cells are obtained from the original screening ofcell lines. These may be Holstein or different lines than those usedabove. This allows crossing while maintaining as much genetic variationin the herd as possible. Introduction of HACs into cell lines andselection of positive cell lines is then effected. Selected cell linesare used for nuclear transfer and calves are produced. Starting at 12months of age semen and eggs are collected, fertilized, and transferredinto recipient animals. Cell samples are taken for DNA marker analysisand karyotyping. Beginning at birth, blood samples are taken andanalyzed for the presence of human Ig proteins.

As indicated above, HACs are also optionally transferred into Homo H/Lcell lines using the procedures developed in the above experiments.

The following examples are additionally provided as furtherexemplification of the invention.

Exemplary Procedures for Insertion of HACs

Additional experiments were carried out to demonstrate that xenogenous(e.g., human) immunoglobulin heavy chain (mu) and lambda light chain maybe produced by a bovine host, either alone or in combination. Inaddition, these experiments demonstrated that the human immunoglobulinchains were rearranged and that polyclonal sera was obtained. In theseprocedures, immunoglobulin-expressing genes were introduced into bovinefibroblasts using human artificial chromosomes. The fibroblasts werethen utilized for nuclear transfer, and fetuses were obtained andanalyzed for antibody production. These procedures and results aredescribed in more detail below.

HAC Constructs The human artificial chromosomes (HACs) were constructedusing a previously described chromosome-cloning system (Kuroiwa et al.,Nature Biotech. 18: 1086-1090, 2000). Briefly, for the construction ofΔHAC, the previously reported human chromosome 22 fragment (hChr22)containing a loxP sequence integrated at the HCF2 locus was truncated atthe AP000344 locus by telomere-directed chromosomal truncation (Kuroiwaet al., Nucleic Acid Res., 26: 3447-3448, 1998). Next, cell hybrids wereformed by fusing the DT40 cell clone containing the above hChr22fragment (hCF22) truncated at the AP000344 locus with a DT40 cell clone(denoted “R clone”) containing the stable and germline-transmittablehuman minichromosome SC20 vector. The SC20 vector was generated byinserting a loxP sequence at the RNR2 locus of the S20 fragment. TheSC20 fragment is a naturally-occurring fragment derived from humanchromosome 14 that includes the entire region of the human Ig heavychain gene (Tomizuka et al., Proc. Natl. Acad. Sci. USA 97:722, 2000).The resulting DT40 cell hybrids contained both hChr fragments. The DT40hybrids were transfected with a Cre recombinase-expression vector toinduce Cre/loxP-mediated chromosomal translocation between hCF22 and theSC20 vector. The stable transfectants were analyzed using nested PCR toconfirm the cloning of the 2.5 megabase hChr22 region, defined by theHCF2 and AP000344 loci, into the loxP-cloning site in the SC20 vector.The PCR-positive cells which were expected to contain ΔHAC were thenisolated by FACS sorting based on the fluorescence of the encoded greenfluorescent protein. Fluorescent in situ hybridization (FISH) analysisof the sorted cells was also used to confirm the presence of ΔHAC, whichcontains the 2.5 megabase hChr22 insert.

Similarly, ΔΔHAC was also constructed using this chromosome-cloningsystem. The hChr22 fragment was truncated at the AP000344 locus, andthen the loxP sequence was integrated into the AP000553 locus byhomologous recombination in DT40 cells. The resulting cells were thenfused with the R clone containing the SC20 minichromosome vector. Thecell hybrids were transfection with a Cre-expression vector to allowCre/loxP-mediated chromosomal translocation. The generation of ΔΔHAC,which contains the 1.5 megabase hChr22 insert, defined by the AP000553and AP000344 loci, was confirmed by PCR and FISH analyses.

The functionality of ΔHAC and ΔΔHAC in vivo was assessed by thegeneration of chimeric mice containing these HACs. These HACs wereindividually introduced into mouse embryonic stem (ES) cells, which werethen used for the generation of chimeric mice using standard procedures(Japanese patent number 2001-142371; filed May 11, 2000). The resultingmice had a high degree of chimerism (85-100% of coat color),demonstrating a high level of pluripotency of the ES cells containingthese HACs and the mitotic stability of these HACs in vivo. Furthermore,ΔHAC was transmitted through the germline of the ΔHAC chimeric mouse tothe next offspring, demonstrating the meiotic stability of this HAC.

Chicken DT40 cells retaining these HACs have been deposited under theBudapest treaty on May 9, 2001 in the International Patent OrganismDepository, National Institute of Advanced Industrial Science andTechnology, Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken, 305-8566 Japan. The depository numbers are as follows: ΔHAC(FERM BP-7582), ΔΔHAC (FERM BP-7581), and SC20 fragment (FERM BP-7583).Chicken DT40 cells retaining these HACs have also been deposited in theFood Industry Research and Development Institute (FIRDI) in Taiwan. Thedepository numbers and dates are as follows: ΔHAC (CCRC 960144; Nov. 9,2001), ΔΔHAC (CCRC 960145; Nov. 9, 2001), and SC20 fragment (the cellline was deposited under the name SC20 (D); CCRC 960099; Aug. 18, 1999).

The 2.5 megabase (Mb) hChr22 insert in ΔHAC is composed of the followingBAC contigs, which are listed by Genbank accession number: AC002470,AC002472, AP000550, AP000551, AP000552, AP000556, AP000557, AP000558,AP000553, AP000554, AP000555, D86995, D87019, D87012, D88268, D86993,D87004, D87022, D88271, D88269, D87000, D86996, D86989, D88270, D87003,D87018, D87016, D86999, D87010, D87009, D87011, D87013, D87014, D86991,D87002, D87006, D86994, D87007, D87015, D86998, D87021, D87024, D87020,D87023, D87017, AP000360, AP00361, AP000362, AC000029, AC000102, U07000,AP000343, and AP000344. The 1.5 Mb hChr22 insert in ΔΔHAC is composed ofthe following BAC contigs: AP000553, AP000554, AP000555, D86995, D87019,D87012, D88268, D86993, D87004, D87022, D88271, D88269, D87000, D86996,D86989, D88270, D87003, D87018, D87016, D86999, D87010, D87009, D87011,D87013, D87014, D86991, D87002, D87006, D86994, D87007, D87015, D86998,D87021, D87024, D87020, D87023, D87017, AP000360, AP00361, AP000362,AC000029, AC000102, U07000, AP000343, and AP000344 (Dunham et al, Nature402:489-499, 1999).

Generation of Bovine Fetal Fibroblasts To generate bovine fetalfibroblasts, day 45 to 60 fetuses were collected from disease-testedHolstein or Jersey cows housed at Trans Ova (Iowa), in which thepedigree of the male and female parents were documented for threeconsecutive generations. The collected fetuses were shipped on wet iceto Hematech's Worcester Molecular Biology Division for the generation ofprimary fetal fibroblasts. Following arrival, the fetus(es) weretransferred to a non-tissue culture grade, 100 mm plastic petri dish ina tissue culture hood. Using sterile forceps and scissors, theextraembryonic membrane and umbilical cord were removed from the fetus.After transferring the fetus to a new plastic petri dish, the head,limbs and internal organs were removed. The eviscerated fetus wastransferred to a third petri dish containing approximately 10 ml offetus rinse solution composed of: 125 ml 1× Dulbecco's-PBS (D-PBS) withCa²⁺ and Mg²⁺ (Gibco-BRL, cat #.14040); 0.5 ml Tylosine Tartrate (8mg/ml, Sigma, cat #.T-3397); 2 ml Penicillin-Streptomycin (Sigma, cat#.P-3539); and 1 ml of Fungizone (Gibco-BRL, cat #.15295-017) (mixed andfiltered through a 0.2 μm nylon filter unit [Nalgene, cat #.150-0020).

The fetus was washed an additional three times with the fetus rinsesolution to remove traces of blood, transferred to a 50 ml conicaltissue culture tube, and finely minced into small pieces with a sterilescalpel. The tissue pieces were washed once with 1×D-PBS without Ca²⁺and Mg²⁺ (Gibco-BRL, cat #.14190). After the tissue pieces settled tothe bottom of the tube, the supernatant was removed and replaced withapproximately 30 ml of cell dissociation buffer (Gibco-BRL, cat#.13151-014). The tube was inverted several times to allow mixing andincubated at 38.5° C./5% CO₂ for 20 minutes in a tissue cultureincubator. Following settling of the tissue to the bottom of the tube,the supernatant is removed and replaced with an equivalent volume offresh cell dissociation buffer. The tissue and cell dissociation buffermixture was transferred to a sterile, 75 ml glass trypsinizing flask(Wheaton Science Products, cat #.355393) containing a 24 mm,round-ended, spin bar. The flask was transferred to a 38.5° C./5% CO₂tissue culture incubator, positioned on a magnetic stir plate, andstirred at a sufficient speed to allow efficient mixing forapproximately 20 minutes. The flask was transferred to a tissue culturehood; the tissue pieces allowed to settle, followed by removal of thesupernatant and harvesting of the dissociated cells by centrifugation at1,200 rpm for five minutes. The cell pellet was re-suspended in a smallvolume of complete fibroblast culture media composed of: 440 ml alphaMEM (BioWhittaker, cat #.12-169F); 50 ml irradiated fetal bovine serum;5 ml GLUTAMAX-I supplement (Gibco-BRL, cat #.25050-061); 5 mlPenicillin-Streptomycin (Sigma, cat #.P-3539); 1.4 ml 2-mercaptoethanol(Gibco-BRL, cat #.21985-023) (all components except the fetal bovineserum were mixed were filtered through 0.2 μm nylon filter unit[Nalgene, cat #.151-4020]), and stored on ice. The dissociation processwas repeated three additional times with an additional 30 ml of celldissociation solution during each step. Cells were pooled; washed incomplete fibroblast media; passed sequentially through 23 and 26 gaugeneedles, and finally through a 70 μm cell strainer (B-D Falcon, cat#.352350) to generate a single cell suspension. Cell density andviability were determined by counting in a hemacytometer in the presenceof trypan blue (0.4% solution, Sigma, cat #.T-8154).

Primary fibroblasts were expanded at 38.5° C./5% CO₂ in completefibroblast media at a cell density of 1×10⁶ viable cells per T75 cm²tissue culture flask. After 3 days of culture or before the cellsreached confluency, the fibroblasts were harvested by rinsing the flaskonce with 1×D-PBS (without Ca²⁺ and Mg²⁺) and incubating with 10 ml ofcell dissociation buffer for 5 to 10 minutes at room temperature.Detachment of cells was visually monitored using an inverted microscope.At this step, care was taken to ensure that cell clumps weredisaggregated by pipetting up-and-down. After washing and quantitation,the dissociated fibroblasts were ready for use in gene targetingexperiments. These cells could also be cryopreserved for long-termstorage.

Introduction of HACs into Bovine Fetal Fibroblasts ΔHAC and ΔΔHAC weretransferred from the DT40 cell hybrids to Chinese hamster ovary (CHO)cells using microcell-mediated chromosome transfer (MMCT) (Kuroiwa etal. Nature Biotech. 18: 1086-1090, 2000). The CHO clone containing ΔHAC(“D15 clone”) was cultured in F12 (Gibco) medium supplemented with 10%FBS (Gibco), 1 mg/ml of G418, and 0.2 mg/ml of hygromycin B at 37° C.and 5% CO₂. The D15 clone was expanded into twelve T25 flasks. When theconfluency reached 80-90%, colcemid (Sigma) was added to the medium at afinal concentration of 0.1 μg/ml. After three days, the medium wasexchanged with DMEM (Gibco) supplemented with 10 μg/ml of cytochalacin B(Sigma). The flasks were centrifuged for 60 minutes at 8,000 rpm tocollect microcells. The microcells were purified through 8, 5, and 3-μmfilters (Costar) and then resuspended in DMEM medium. The microcellswere used for fusion with bovine fibroblasts as described below.

Bovine fetal fibroblasts were cultured in α-MEM (Gibco) mediumsupplemented with 10% FBS (Gibco) at 37° C. and 5% CO₂. The fibroblastswere expanded in a T175 flask. When the confluency reached 70-80%, thecells were detached from the flask with 0.05% trypsin. The fibroblastcells were washed twice with DMEM medium and then overlayed on themicrocell suspension. After the microcell-fibroblast suspension wascentrifuged for five minutes at 1,500 rpm, PEG1500 (Roche) was added tothe pellet according to the manufacturer's protocol to enable fusion ofthe microcells with the bovine fibroblasts. After fusion, the fusedcells were plated into six 24-well plates and cultured in α-MEM mediumsupplemented with 10% FBS for 24 hours. The medium was then exchangedwith medium containing 0.7 mg/ml of G418. After growth in the presenceof the G418 antibiotic for about two weeks, the G418 resistant, fusedcells were selected. These G418-resistant clones were used for nucleartransfer, as described below.

Similarly, ΔΔHAC from the CHO clone C13 was transferred into bovinefetal fibroblasts by means of MMCT. The selected G418-resistant cloneswere used for nuclear transfer.

Nuclear Transfer Activation and Embryo Culture The nuclear transferprocedure was carried out essentially as described earlier (Cibelli etal., Science 1998: 280:1256-1258). In vitro matured oocytes wereenucleated about 18-20 hours post maturation (hpm) and chromosomeremoval was confirmed by bisBenzimide (Hoechst 33342, Sigma) labelingunder UV light. These cytoplast-donor cell couplets were fused, by usingsingle electrical pulse of 2.4 kV/cm for 20 μsec (Electrocellmanipulator 200, Genetronics, San Diego, Calif.). After 3-4 hrs, arandom sub-set of 25% of the total transferred couplets was removed, andthe fusion was confirmed by bisBenzimide labeling of the transferrednucleus. At 30 hpm reconstructed oocytes and controls were activatedwith calcium ionophore (5 μM) for 4 minutes (Cal Biochem, San Diego,Calif.) and 10 μg Cycloheximide and 2.5 μg Cytochalasin D (Sigma) in ACMculture medium for 6 hours as described earlier (Lin et al., Mol.Reprod. Dev. 1998: 49:298-307; Presicce et al., Mol. Reprod. Dev.1994:38:380-385). After activation eggs were washed in HEPES bufferedhamster embryo culture medium (HECM-Hepes) five times and placed inculture in 4-well tissue culture plates containing irradiated mousefetal fibroblasts and 0.5 ml of embryo culture medium covered with 0.2ml of embryo tested mineral oil (Sigma). Twenty five to 50 embryos wereplaced in each well and incubated at 38.5° C. in a 5% CO₂ in airatmosphere. On day four 10% FCS was added to the culture medium.

Embryo Transfer Day 7 and 8 nuclear transfer blastocysts weretransferred into day 6 and 7 synchronized recipient heifers,respectively. Recipient animals were synchronized using a singleinjection of Lutalyse (Pharmacia & Upjohn, Kalamazoo, Mich.) followed byestrus detection. The recipients were examined on day 30 and day 60after embryo transfer by ultrasonography for the presence of conceptusand thereafter every 30 days by rectal palpation until 270 days. Theretention of a HAC in these bovine fetuses is summarized in Table 1 andis described in greater detail in the sections below. TABLE 1 Summary ofHAC retention in bovine fetuses Recip/ HAC Cell Fetus NT RecoveryRetention HAC Clone No. Date Date Fetal Age H L ΔΔ 4-12 5580 2/14 4/1358 + + ΔΔ 2-14 5848 2/15 4/13 57 − − ΔΔ 4-12 5868A 2/14 6/13 119  + + ΔΔ4-12 5868B 2/14 6/13 119  + + ΔΔ 4-12 5542A 2/14 5/16 91 + + ΔΔ 4-125542B 2/14 5/16 91 + + ΔΔ 4-12 5174 2/14 5/16 91 (abnormal) nd nd ΔΔ4-12 6097 2/14 Remains 160 (7/24) nd nd Δ 4-8 6032 1/31 3/30 58 + + Δ2-13 5983 2/2 3/30 56 − − Δ 4-2 5968 2/2 3/30 56 + + Δ 2-22 6045 2/23/30 56 + + Δ 4-8 5846 1/31 4/20 79 − − Δ 2-13 6053 2/2 4/27 84 + − Δ4-2 5996 2/1 4/20 77 + −

Introduction of a HAC containing a fragment of human chromosome #14 TheSC20 fragment, a human chromosome #14 fragment (“hchr.14fg”, containingthe Ig heavy chain gene), was introduced into fetal fibroblast cells insubstantially the same manner as described above. Any other standardchromosome transfer method may also be used to insert this HAC oranother HAC containing a human Ig gene into donor cells. The resultingdonor cells may be used in standard nuclear transfer techniques, such asthose described above, to generate transgenic ungulates with the HAC.

The pregnancy status of the 28 recipients to whom cloned embryos weretransferred from cells containing the hchr.14fg was checked byultrasonography. The results are summarized in Table 2.

If desired, a cell from the resulting HAC fetus or HAC offspring can beused in a second round of nuclear transfer to generate additional clonedoffspring. Cells from the initial HAC fetus or HAC offspring may also befrozen to form a cell line to be used as a source of donor cells for thegeneration of additional HAC ungulates. TABLE 2 Pregnancy at 40 daysusing donor cells containing hchr.14fg No of recips Pregnancy at 40 daysClone ID transferred (%) 2-1 08 03 (38) 4-2 10 00 (00) 4-1 05 00 (00)4-1 03 01 (33) 2-1 02 01 (50) Total 28 05 (18)

The pregnancy rates were lower than anticipated. This is believed to beattributable to extremely abnormally hot weather during embryo transfer.

As illustrated in FIG. 27, pregnancy rates for HAC carrying embryosappear to be equivalent to non-transgenic cloned pregnancies. Onerecipient carrying a ΔΔHAC calf gave birth recently to a live healthycalf. Others will be born over the next several months.

Demonstration of Rearrangement and Expression of Human Heavy Chain Locusin a ΔHAC Bovine Fetus

Cloned ΔHAC-transgenic bovine fetuses were removed at variousgestational days and analyzed for the presence, rearrangement, andexpression of the human immunoglobulin loci. Analysis of genomic DNA andcDNA obtained by RT-PCR from spleen and nonlymphoid tissues (liver andbrain) of one of these fetuses indicated the presence, rearrangement,and expression of the ΔHAC.

Presence of Human Heavy and/or Light Chain in ΔHAC Fetuses To determinewhether the human heavy and light chains were retained in ΔHAC fetuses,liver DNA was isolated from ΔHAC fetuses and analyzed by PCR for thepresence of genomic DNA encoding human heavy and light chains.

For the detection of genomic heavy chain DNA, the following primers wereused: VH3-F 5′-AGTGAGATAAGCAGTGGATG-3′ (SEQ ID NO: 1) and VH3-R5′-CTTGTGCTACTCCCATCACT-3′ (SEQ ID NO: 2). The primers used fordetection of lambda light chain DNA were IgL-F5′-GGAGACCACCAAACCCTCCAAA-3′ (SEQ ID NO: 3) and IgL-R5′-GAGAGTTGCAGAAGGGGTYGACT-3′ (SEQ ID NO: 4). The PCR reaction mixturescontained 18.9 μl water, 3 μl of 10×Ex Taq buffer, 4.8 μl of dNTPmixture, 10 pmol forward primer and 10 pmol of reverse primer, 1 μl ofgenomic DNA, and 0.3 μl of Ex Taq. Thirty-eight cycles of PCR wereperformed by incubating the reaction mixtures at the followingconditions: 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, 56° C. for 30 seconds, and 72° C. for 30 seconds.

As shown in FIG. 5, fetuses #5968, 6032 and 6045 each contained bothhuman heavy chain (μ) and light chain (

) loci. Fetus #5996 contained only the human heavy chain locus. Fetus#5983 did not contain the human heavy chain and may not have containedthe human light chain. Fetus #5846 did not contain either humansequence. Thus, fetuses #5983 and 5846 may not have retained the HAC.These results suggested that ΔHAC can be stably retained up togestational day 58 in bovines.

Presence of Human Cmu Exons in ΔHAC Fetus #5996 Primers specific for amRNA transcript including portions of Cmu 3 and Cmu 4 were used todetermine whether ΔHAC was present and expressing transcripts encodingthe constant region of the human mu locus of fetus #5996.

For this RT-PCR analysis of the genomic constant region of the human muheavy chain, primers “CH3-F1” (5′accacctatgacagcgtgac-3′, SEQ ID NO: 5)and “CH4-R2” (5′-gtggcagcaagtagacatcg-3′, SEQ ID NO: 6) were used togenerate a RT-PCR product of 350 base pairs. This PCR amplification wasperformed by an initial denaturing incubation at 95° C. for fiveminutes. Then, 35 cycles of denaturation, annealing, and amplificationwere performed by incubation at 95° C. for one minute, 59° C. for oneminute, and 72° C. for two minutes. Then, the reaction mixtures wereincubated at 72° C. for 10 minutes. Rearranged bovine heavy chain wasdetected using primers I7L and P9, as described below (FIG. 7). As aninternal control, levels of GAPDH RNA was detected using primers “GAPDHforward” (5′-gtcatcatctctgccccttctg-3′, SEQ ID NO: 7) and “GAPDHreverse” (5′-aacaacttcttgatgtcatcat-3′, SEQ ID NO: 8). For thisamplification of GAPDH RNA, samples were incubated at 95° C. for fiveminutes, followed by 35 cycles of incubation at 95° C. for one minute,55° C. for one minute, and 72° C. for two minutes. Then, the mixtureswere incubated at 72° C. for seven minutes.

This analysis showed that RT-PCR analysis of the spleen of fetus #5996produced a band (lane 3) matching the amplification products generatedusing control human spleen cDNA (lane 4) and cDNA obtained from a ΔHACchimeric mouse (lane 5) (FIG. 6). No such band was detected innonlymphoid tissues: bovine liver (lane 1) or bovine brain (lane 2). Thecapacity of these tissues to support RT-PCR was shown by the successfulamplification of the housekeeping gene, GADPH, in both liver (lane 10 ofFIG. 6) and brain (lane 6 of FIG. 7).

Rearrangement of Bovine Heavy Chain Locus by 77 Gestational Days TheΔHAC fetus #5996 was tested to determine whether it had undergone thedevelopmental processes necessary for the expression and activation ofthe recombination system required for immunoglobulin heavy chain locusrearrangement. For this analysis, standard RT-PCR analysis was performedto detect the presence of mRNA transcripts encoding mu-VHrearrangements. RNA isolated from the spleen, liver, and brain of fetus#5996 was analyzed by RT-PCR using primers “17L”(5′-ccctcctctttgtgctgtca-3′, SEQ ID NO: 9) and “P9”(5′-caccgtgctctcatcggatg-3′, SEQ ID NO: 10). The PCR reaction mixtureswere incubated at 95° C. for 3 minutes, and then 35 cycles ofdenaturation, annealing, and amplification were performed using thefollowing conditions: 95° C. for one minute, 58° C. for one minute, and72° C. for two minutes. The reaction mixture was then incubated at 72°C. for 10 minutes.

Lane 5 of FIG. 7 shows that a product of the size expected foramplification of a rearranged bovine heavy chain (450 base pairs) wasobtained. This product migrated to a position equivalent to that of acontrol bovine Cmu heavy chain cDNA known to contain sequencescorresponding to rearranged bovine heavy chain transcripts (lane 7). Asexpected, the rearranged heavy chain was expressed in the spleen (lane5), but absent from the brain (lane 2) and liver (lane 3) at this pointin development.

Rearrangement and Expression of the Human Heavy Chain locus in the ΔHACFetus #5996 The rearrangement and expression of the human heavy chainlocus was demonstrated by the amplification of a segment of DNAincluding portions of Cmu and VH regions. Primers specific for RNAtranscripts including portions of Cmu (Cmu1) and VH (VH3-30) were usedto determine if RNA transcripts containing rearranged human Cmu-VDJsequences were present (FIG. 8).

For this RT-PCR analysis, primers “Cmu1” (5′-caggtgcagctggtggagtctgg-3′,SEQ ID NO: 11) and “VH3-30” (5′caggagaaagtgatggagtc-3′, SEQ ID NO: 12)were used to produce a RT-PCR product of 450 base pairs. This RT-PCR wasperformed by incubating reaction mixtures at 95° for 3 minutes, followedby 40 cycles of incubation at 95° for 30 minutes, 69° for 30 minutes,and 72° for 45 minutes, and one cycle of incubation at 72° for 10minutes. This RT-PCR product was then reamplified with the same primersby one cycle of incubation at 95° C. for three minute, 40 cycles ofincubation at 95° C. for one minute, 59° C. for one minute, 72° C. forone minute, and one cycle of incubation at 72° C. for 10 minutes. As aninternal control, RT-PCR amplification of GAPDH was performed asdescribed above.

The gel in FIG. 8 shows that RT-PCR analysis of the spleen from fetus#5996 produced a band (lane 5) matching the amplification productsgenerated using human spleen cDNA (lane 4) or ΔHAC chimeric mouse spleencDNA (lane 1). No such band was detected in bovine liver (lane 2) orbovine brain (lane 3). As a positive control, amplification of GADPH RNA(lanes 8 and 9) showed the capacity of these tissues to support RT-PCR.

Rearrangement and expression of the human heavy chain region in fetus#5996 was also demonstrated by RT-PCR analysis using primers CH3-F3(5′-GGAGACCACCAAACCCTCCAAA-3′, SEQ ID NO: 13) and CH4-R2(5′-GTGGCAGCAAGTAGACATCG-3′, SEQ ID NO: 14). These PCR reaction mixturescontained 18.9 μl water, 3 μl of 10×Ex Taq buffer, 4.8 μl of dNTPmixture, 10 pmol forward primer, 10 pmol of reverse primer, 1 μl ofcDNA, and 0.3 μl of Ex Taq. Forty PCR cycles were performed byincubating the reaction mixtures under the following conditions: 85° C.for three minutes, 94° C. for one minute, 98° C. for 10 seconds, 60° C.for 30 seconds, and 72° C. for 30 seconds.

As shown in lanes 6 and 7 of FIG. 9, an amplified sequence from thespleen of fetus #5996 was the same size as the spliced constant regionfragments from the two positive controls: a sample from a human spleen(lane 8) and a ΔHAC chimeric mouse spleen (lane 9). As expected, thenegative controls from a normal mouse spleen and a bovine spleen did notcontain an amplified sequence (lanes 1 and 2). Samples from the liverand brain of fetus #5996 did not contain an amplified spliced sequenceof the same size as the spliced human mu heavy chain constant regionfragments but did contain a amplified sequence of an unspliced genomicfragment derived from genomic DNA contaminating the RNA sample (lanes 3,4, and 5).

VDJ Rearrangement of the Human Heavy Chain Locus in a ΔHAC Fetus RT-PCRanalysis was also performed to further demonstrate VDJ rearrangement inthe heavy chain locus in ΔHAC fetus #5996. Nested RT-PCR was performedusing primer Cmu-1 (5′-CAGGAGAAAGTGATGGAGTC-3′, SEQ ID NO: 15) for thefirst reaction, primer Cmu-2 (5′-AGGCAGCCAACGGCCACGCT-3′, SEQ ID NO: 16)for the second reaction, and primer VH3-30.3(5′-CAGGTGCAGCTGGTGGAGTCTGG-3′, SEQ ID NO: 17) for both reactions. TheRT-PCR reaction mixtures contained 18.9 μl water, 3 μl of 10×Ex Taqbuffer, 4.8 μl of dNTP mixture, 10 pmol forward primer, 10 pmol ofreverse primer, 1 μl of cDNA, and 0.3 μl of Ex Taq. The RT-PCR wasperformed using 38 cycles under the following conditions for the firstreaction: 85° C. for three minutes, 94° C. for one minute, 98° C. for 10seconds, 65° C. for 30 seconds, and 72° C. for 30 seconds. For thesecond reaction, 38 cycles were performed under the followingconditions: 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, 65° C. for 30 seconds, and 72° C. for 30 seconds usingprimers VH3-30.3 and Cmu-2 (5′-AGGCAGCCAACGGCCACGCT-3′, SEQ ID NO: 16).

As shown in lanes 6 and 7 of FIG. 10, RT-PCR analysis of the spleen offetus #5996 produced a heavy chain band of the same size as the positivecontrols in lanes 8 and 9. Samples from the liver and brain of fetus#5996 contained some contaminating rearranged DNA (lanes 3 and 5). Thenegative controls in lanes 1 and 2 produced bands of the incorrect size.

Verification Of ΔHAC Rearrangement By Sequencing The cDNA obtained byreverse transcription of RNA from the spleen of the ΔHAC fetus #5996 wasamplified with primers specific for rearranged human mu and run on anagarose gel. The band produced by amplification with the Cmu1-VH3-30primer pair was excised from the gel. The amplified cDNA was recoveredfrom the band and cloned. DNA from a resulting clone that wasPCR-positive for rearranged human mu was purified and sequenced (FIG.11A).

The sequence from this ΔHAC fetus is greater than 95% homologous to over20 known human heavy chain sequences. For example, the mu chain of ahuman anti-pneumococcal antibody is 97% homologous to a region of thissequence (FIG. 11B).

Additional sequences from rearranged human heavy chains were alsoobtained by RT-PCR analysis of the spleen of fetus #5996 using primersCmu-1 and VH3-30.3, followed by reamplification using primers Cmu-2 andVH3-30.3. The RT-PCR products were purified using CHROMA SPIN column(CLONETECH) and cloned into the pCR2.1 TA-cloning vector (Invitrogen)according to manufacturer's protocol. The Dye Terminator sequencereaction (ABI Applied System) was performed in a 10 μl volume reactionmixture composed of BigDye Terminator reaction mixture (3 μl), templateplasmid (200 ng), and the Cmu-2 primer (1.6 pmol). The sequencingreaction was performed using a ABI 3700 sequencer. For this analysis,twenty-five cycles were conducted under the following conditions: 96° C.for one minute, 96° C. for 10 seconds, 55° C. for five seconds, and 60°C. for four minutes.

At least two rearranged human heavy chain transcripts were identified,which were VH3-11/D7-27/JH3/Cμ and VH3-33/D6-19/JH2/Cμ (FIGS. 12A and12B). These results demonstrate that VDJ rearrangement of the human muheavy chain locus occurs in the ΔHAC in the spleen of fetus #5996. Theidentification of more than one rearranged heavy chain sequence from thesame fetus also demonstrates the ability of ΔHAC fetuses to generatediverse human immunoglobulin sequences.

Rearrangement and Expression of Human Heavy and Light Chain Loci inΔΔHAC Fetus

Cloned fetuses derived from bovine fetal fibroblasts transchromosomalfor the ΔΔHAC were removed from recipient cows at various gestationaldays. The fetuses were analyzed for the presence and rearrangement ofthe HAC-borne human immunoglobulin heavy and lambda light chain loci.Studies of genomic DNA from these tissues indicated the presence of thehuman immunoglobulin heavy and light chains in some of the fetuses.Examination of cDNA derived from the spleens of these fetuses indicatedrearrangement and expression of the immunoglobulin heavy and light chainloci in some of these fetuses. FACS analysis also demonstrated theexpression of human lambda light chain protein on the surface of spleniclymphocytes in two of the fetuses.

Presence of Human Heavy and Light Chain Loci in ΔΔHAC Fetuses Todetermine whether ΔΔHAC fetuses retained the human heavy and light chainloci, PCR analysis was performed on genomic DNA from the liver of 58 dayfetus #5580, 57 day fetus #5848, and 91 day fetuses #5442A and 5442B.The PCR primers used for detection of the heavy chain loci were VH3-F(5′-AGTGAGATAAGCAGTGGATG-3′, SEQ ID NO: 18) and VH3-R(5′-CTTGTGCTACTCCCATCACT-3′, SEQ ID NO: 19), and the primers used forthe detection of the light chain were IgL-F(5′-GGAGACCACCAAACCCTCCAAA-3′, SEQ ID NO: 20) and IgL-R(5′-GAGAGTTGCAGAAGGGGTYGACT-3′, SEQ ID NO: 21). The PCR reactionmixtures contained 18.9 μl water, 3 ul of 10×Ex Taq buffer, 4.8 μl ofdNTP mixture, 10 pmol forward primer, 10 pmol of reverse primer, 1 μl ofgenomic DNA, and 0.3 ul of Ex Taq. Thirty-eight PCR cycles wereperformed as follows: 85° C. for three minutes, 94° C. for one minute,98° C. for 10 seconds, 56° C. for 30 seconds, and 72° C. for 30 seconds(FIGS. 13 and 14).

As illustrated in FIGS. 13 and 14, positive control 58 day fetus #5580contained both human heavy and light chain immunoglobulin loci.Additionally, the 91 day fetuses #5442A and 5442B also contained bothheavy and light chain loci (FIG. 14). In contrast, fetus #5848 did notcontain either human loci and may not have contained ΔΔHAC. Theseresults suggested that ΔΔHAC can be stably retained up to gestationalday 91 in bovine.

Rearrangement and Expression of Human Heavy Chain Locus in ΔΔHAC Fetus#5442A RT-PCR was used to detect expression of rearranged human heavychain RNA transcripts in ΔΔHAC fetus #5542A. The RT-PCR primers usedwere CH3-F3 (5′-GGAGACCACCAAACCCTCCAAA-3′, SEQ ID NO: 22) and CH4-R2(5′-GAGAGTTGCAGAAGGGGTGACT-3′, SEQ ID NO: 23). The RT-PCR reactionmixtures contained 18.9 μl water, 3 μl of 10×Ex Taq buffer, 4.8 μl ofdNTP mixture, 10 pmol forward primer, 10 pmol of reverse primer, 1 μl ofcDNA, and 0.3 μl of Ex Taq. Forty cycles of RT-PCR cycles were performedas follows: 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds.

Lanes 4 and 5 of FIG. 15 contained amplified spliced mu heavy chainconstant region sequences from the spleen of fetus #5442A that aresimilar in size to that of the positive control samples. These resultsindicate that fetus #5442A expressed a rearranged mu heavy chaintranscript in its spleen. Faint bands were also seen in the region ofthe unspliced genomic sequence, which are amplified from genomic DNAcontaminated in the RNA sample. Control samples from the liver and brainof fetus #5442A did not produce a band of the size expected for anamplified rearranged heavy chain sequence.

Rearrangement and Expression of Human Heavy Chain Locus in ΔΔHAC Fetus#5868A RT-PCR was used to detect expression of rearranged human heavychain RNA transcripts in the spleen of a ΔΔHAC fetus at 119 gestationaldays (fetus #5868A). The primers used for this analysis were VH30-3(5′-caggtgcagctggtggagtctgg-3′, SEQ ID NO: 24) and CM-1(5′-caggagaaagtgatggagtc-3′, SEQ ID NO: 25). Additionally, primers“GAPDH up” (5′-gtcatcatctctgccccttctg-3′, SEQ ID NO: 26) and “GAPDHdown” (5′-aacaacttcttgatgtcatcat-3′, SEQ ID NO: 27) were used to amplifyGAPDH control transcripts. For this PCR analysis, the reaction mixturewas incubated at 95° C. for five minutes and then multiple cycles ofdenaturation, annealing, and amplification were performed by incubationat 95° C. for one minute, 58° C. for one minute, and 72° C. for twominutes. Then, the mixture was incubated at 72° C. for 10 minutes.

Lane 3 of FIG. 16 contains the RT-PCR product produced from thisanalysis of ΔΔHAC fetus #5868A. This RT-PCR product was the sizeexpected for the amplification of a rearranged human heavy chain (470base pairs) and migrated to the same position in the gel as the controlcDNA known to contain sequences corresponding to rearranged human heavychain transcripts. As controls, both ΔΔHAC fetus #5868A fetal spleencDNA and normal bovine cDNA samples generated a product when amplifiedwith GAPDH primers, demonstrating the capacity of the cDNA to supportamplification (lanes 7 and 8).

Rearrangement and Expression of Human Lambda Locus in ΔΔHAC Fetuses#5442A and 5442B Primers specific for amplification of a transcriptincluding portions of human lambda were used to detect RNA transcriptsfrom a rearranged human lambda light chain locus.

For the RT-PCR analysis shown in FIG. 17, an equimolar mixture ofprimers Cλ1 (5′-GGGAATTCGGGTAGAAGTTCACTGATCAG-3′, SEQ ID NO: 28), Cλ2-3(5′-GGGAATTCGGGTAGAAGTCACTTATGAG-3′, SEQ ID NO: 29), and Cλ7(5′-GGGAATTCGGGTAGAAGTCACTTACGAG-3′, SEQ ID NO: 30) was used with primerVλ1 LEA1 (5′-CCCAAGCTTRCCKGSTYYCCTCTCCTC-3′, SEQ ID NO: 31). The RT-PCRreaction mixtures contained 18.9 μl water, 3 μl of 10×Ex Taq buffer, 4.8μl of dNTP mixture, 10 pmol forward primer, 10 pmol of reverse primer, 1μl of cDNA and 0.3 μl of Ex Taq. The RT-PCR conditions were as follows:40 cycles of 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, 60° C. for 30 seconds, and 72° C. for one minute.

As shown in FIG. 18, this RT-PCR analysis was also performed using anequimolar mixture of primers Vλ3LEA1(5′-CCCCCAAGCTTGCCTGGACCCCTCTCTGG-3′; SEQ ID NO:32), Vλ3JLEAD(5′-ATCGGCAAAGCTTGGACCCCTCTCTGGCTCAC-3′, SEQ ID NO: 33), VλBACK4(5′-CCCCCAAGCTTCTCGGCGTCCTTGCTTAC-3′, SEQ ID NO: 34) and an equimolarmixture of primers Cλ1 (5′-GGGAATTCGGGTAGAAGTTCACTGATCAG-3′, SEQ ID NO:35) Cλ2-3 (5′-GGGAATTCGGGTAGAAGTCACTTATGAG-3′, SEQ ID NO: 36) and Cλ7(5′-GGGAATTCGGGTAGAAGTCACTTACGAG-3′, SEQ ID NO: 37). The RT-PCR reactionconditions were the same as those described above for FIG. 7.

Lanes 6 and 7 of FIG. 17 and lanes 4 and 5 of FIG. 18 contained RT-PCRproducts from the spleen of fetus #5442A that are similar in size to thepositive control bands, indicating the presence of rearranged lightchain RNA transcripts in this fetus. The spleen sample from fetus #5442Bproduced very weak bands of the appropriate size which are not visiblein the picture. This RT-PCR product indicates that fetus #5442B alsoexpressed a rearranged light chain immunoglobulin transcript in itsspleen. As expected, samples from the brain of fetuses #5442A and 5442Bdid not express human rearranged lambda light chain transcripts.

Rearrangement and Expression of Human Lambda Locus in ΔΔHAC Fetus #5868ARNA transcripts from a rearranged human lambda light chain locus werealso detected in ΔΔHAC fetus #5868A. For this analysis, primers specificfor amplification of a transcript including portions of human lambdawere used to detect ΔΔHAC-encoded expression of transcripts encodingportions of a rearranged human lambda locus. Primer VL1 LEAI(5′-cccccaagcttRccKgStYYcctctcctc-3′; SEQ ID NO:38) and an equimolarmixture of primers CL1 (5′-gggaattcgggtagaagtcactgatcag-3′; SEQ IDNO:39), CL2-3 (5′-gggaattcgggtagaagtcacttatgag-3′; SEQ ID NO:40), andCL7 (5′-gggaattcgggtagaagtcacttacgag-3′; SEQ ID NO:41) were used forthis analysis. For this RT-PCR reaction, the reaction mixtures wereincubated at 95° C. for 5 minutes and then multiple cycles ofdenaturation, annealing, and amplification were performed by incubationat 95° C. for one minute, 60° C. for one minute, and 72° C. for twominutes. Then, the mixtures were incubated at 72° C. for 10 minutes.

This analysis demonstrated that spleen cDNA from ΔΔHAC #5868A (lane 4 ofFIG. 19) produced a RT-PCR product of the same size as the TC mousespleen cDNA (lane 6) positive control. No such RT-PCR product wasdetected using either brain or liver cDNA from ΔΔHAC #5868A (lanes 2 and3, respectively). The capacity of each of these tissues to supportRT-PCR was shown by successful amplification of the housekeeping gene,GAPDH using primers “GAPDH up” and “GAPDH down” (lanes 8 and 10).

Verification of ΔΔHAC Rearrangement by Sequencing RT-PCR analysis wasperformed on a spleen sample from fetus #5442A using an equimolarmixture of primers Cλ1, Cλ2-3, and Cλ7 with primer Vλ1LEA1, or anequimolar mixture of primers Vλ3LEA1, Vλ3JLEAD, and VλBACK4 and anequimolar mixture of primers Cλ1, Cλ2-3, and Cλ7. The PCR products werepurified using a CHROMA SPIN column (CLONETECH) and cloned into thepCR2.1 TA-cloning vector (Invitrogen), according to manufacturer'sprotocol. The Dye Terminator sequence reaction (ABI Applied System) wascarried out using the Cλ1, Cλ2-3, and Cλ7 primers in an equimolarmixture. Twenty-five cycles were performed at 96° C. for one minute, 96°C. for 10 seconds, 55° C. for five seconds, and 60° C. for four minutes.The 10 μl reaction mixture contained BigDye Terminator reaction mixture(3 μl), template plasmid (200 ng), and the Cλ1, Cλ2-3, and Cλ7 primers(1.6 pmol). The reaction mixture was analyzed using a ABI 3700sequencer.

At least two rearranged human lambda light chain transcripts wereidentified (V1-17/JL3/Cλ and V2-13/JL2/Cλ). These results demonstratethat VJ rearrangement of human lambda light chain genes occurs in theΔΔHAC in the spleen of fetus #5442A (FIGS. 20 and 21).

FACS Analysis of Expression of Human Lambda Light Chain and Bovine HeavyChain in ΔΔHAC Fetus #5442A and 5442B Splenic lymphocytes from ΔΔHACFetus #5442A and 5442B were analyzed for the expression of human lambdalight chain and bovine heavy chain proteins. These cells were reactedwith a phycoerytherin labeled anti-human lambda antibody (FIGS. 22C and22D), a FITC labeled anti-bovine IgM antibody (FIGS. 22D and 22H), or noantibody (FIGS. 22A, 22B, 22E, and 22F) for 20 minutes at 4° C. Cellswere then washed twice with PBS plus 2% FCS and analyzed on a FASCaliburcell sorter. The percent of cells reacting with the antibody wascalculated using the non antibody controls to electronically set thegates. These percentages are displayed beneath each histogram. Fetus#5442A (FIGS. 22A-22D) and fetus #5442B (FIGS. 22E-22H) expressed bothhuman lambda light chain protein and bovine heavy chain protein.

Expression of Human Antibody Protein in HAC Calves

As described above, a novel procedure was developed for producingtranschromosomic (Tc) calves (FIG. 37). This method overcame thelimitations due to the limited life span of only about 35 populationdoublings for primary bove fibroblasts and the requirement for a largeDNA insert to be introduced and maintained in the donor cells. Inparticular, a human artificial chromosome (HAC) vector was used tointroduce both the entire unrearranged human Ig heavy (IgH) and lambdalight chain (Igλ) loci into bovine primary fibroblasts. Selectedfibroblast clones were rejuvenated and expanded by producing clonedfetuses. Cloned fetal cells were selected and recloned to produce fourhealthy, Tc calves that functionally rearranged both heavy and lightchain human Ig loci and produced human polyclonal antibodies. Theseresults demonstrate the feasibility of using HAC vectors for productionof transgenic livestock. More importantly, Tc cattle containing human Iggenes may be used to produce novel human polyclonal therapeutics withapplications ranging from the prevention of antibiotic resistantinfections to combating bioterrorism. This method is described in moredetail below.

HACs were introduced into bovine fetal fibroblasts from CHO clones usingthe MMCT technique described herein. The fibroblasts were placed underselection for the neo gene marker on the HAC vector with G418 (700μg/ml) until colonies began to appear. Complete antibiotic selection andDNA-based screening was avoided at this step to minimize cell divisionsprior to nuclear transfer. Colonies were picked on the basis of growthand morphology and nuclear transfer was carried out as previouslydescribed (Lucier et al., J. Immunol 161:5438-5444, 1998). Because thecells were only useful for nuclear transfer for a few days, finalselection was done after rejuvenation and expansion of cells byproduction of cloned fetuses.

Development to the blastocyst stage ranged from 17 to 21% and pregnancyat 40 days ranged from 22 to 50% with no differences among cell lines.At 56 to 58-days, four ΔHAC and two ΔΔHAC fetuses were recovered andfibroblast cell lines were regenerated and cryopreserved for furtheranalysis and nuclear transfer. Retention of the HACs in the fibroblastlines derived from the fetuses collected at 56 to 58-days and sevenadditional fetuses collected between 77 and 19-days was evaluated byG418-resistance (FIG. 38A) and genomic PCR of IgH and Igλ loci (FIG.38B). Nine of 13 fetuses were resistant to G418 and eight of them showedthe presence of both human IgH and Igλ loci. Three ΔHAC (#5968, #6032and #6045) and one ΔΔHAC (#5580) positive 56 to 58-day fetuses were usedfor recloning and production of offspring. Five positive 77 to 119-dayfetuses were evaluated for expression and rearrangement of the human Igloci by RT-PCR analysis, followed by sequencing of the amplifiedproducts. Human IgH and Igλ genes were expressed (FIG. 38C) in allfetuses and showed evidence of proper V(D)J recombination (FIG. 38D).

Recloned ΔHAC cell lines produced one male (from cell line #6045) and 5female (from cell lines #5968 and #6032) calves from 37 recipients (16%,FIG. 39A). The two calves derived from cell line #6032 died within 48hours after birth. One calf was born from non-regenerated ΔΔHAC cells.All five live calves were healthy and phenotypically normal. Retentionof the HAC was confirmed in all the calves by G418 selection, genomicPCR and fluorescent in situ hybridization (FISH) analyses (FIGS. 39B and39C). Results of FISH analysis indicated that the HAC was retained as anindependent chromosome and the proportion of cells retaining the HAC was78 to 100%. No obvious differences were observed in retention ratesbetween peripheral blood lymphocytes (PBLs, 91%) and fibroblasts (87%)however, donor cell line #6045 may have had a higher retention rate thandonor cell line #5968 (97% and 86%, respectively). Interestingly,retention rate in the Tc bovine may be higher than we previouslyobserved in mouse. To determine whether the human Ig loci wererearranged and expressed in calves as well as in fetuses, we performedRT-PCR analysis on PBLs. We observed the expression of both human IgHand Igλ genes in the PBLs and the diversity of the human IgH and Igλrepertoire was determined by sequence analysis (Table 3). Arepresentative set of the sequences showed a wide utilization ofV_(H)/Vλ, D_(H) and J_(H)/Jλ segments distributed over the loci. In theIgλ transcripts, the frequent utilization of V segments from V_(H)1 andV_(H)3 was observed, which is similar to the usage of V_(H) segments inhuman. Addition of non-germline nucleotides (N-addition), as well asnucleotide deletion, was also observed in both IgH and Igλ transcripts.This produced a high degree of diversification in the thirdcomplementarity determining regions (CDR3s) of both heavy and lightchains. Furthermore, human Ig protein was detected at levels rangingfrom 13 to 258 ng/ml (Ig expression is typically very low toundetectable in newborn calves) in blood samples collected prior tocolostrum feeding in 5 of the seven calves as determined by solid phaseELISA. These data indicate that the HAC transfer can be accomplishedefficiently in primary cells using a recloning strategy and that humanIg genes carried by the HAC can be properly processed and expressed witha high degree of diversity in Tc calves.

The results of this study demonstrate that a combination ofchromosome-cloning, chromosome transfer and somatic cell recloningtechnologies can be used to produce healthy calves retaining a HACvector carrying Mb-sized genomic transgenes. Furthermore, thesetechnologies were used to demonstrate the transfer and retention of theentire loci for both the human IgH and Igλ genes in cattle.Interestingly, both loci were demonstrated to have undergone properprocessing and express functionally rearranged human immunoglobulingenes in a species with substantially different immunophysiology thaneither the human or mouse. This HAC system may be useful for theexpression of a variety of complex human proteins (e.g., hemoglobulin)or large collections of proteins for pharmaceutical applications. Forexample, the Tc calves produced in this study, which retain both thehuman IgH and Igλ loci, are useful for production of human polyclonalantibodies. Human polyclonal antibodies are currently only availablefrom human blood or plasma donors. Consequently, there are limitationsin supply and application of human polyclonal products. Tc cows may behyperimmunized to produce large quantities of novel polyclonaltherapeutics for treatment of a wide variety of human diseases.

Table 3. Repertoire analysis of human immunoglobulin heavy and lambdachain transcripts in cloned Tc calves. Human μ and λ-specific mRNAs wereamplified by RT-PCR, cloned and sequenced. Nucleotide sequences of V(D)Jjunctions of each of 10 independent μ and λ clones are shown, dividedinto V_(H)/Vλ, D_(H), J_(H)/Jλ and N segments, as identified by homologyto published germline sequences (Ig-BLAST). Human μ Nucleotide SequencesV_(H) N D_(H) N J_(H) 6-1 0 D5-24 3 JH3 TACTGTGCA----- AGAGATG AGA-ATGCTTTTGATGTC 3-33 8 D6-13 3 JH4 ATTACTGTGCGA---- AGAACAAAATAGCAGCAGCTGGTAC GAT ----CTTTGACTACT 3-15 4 D6-19 4 JH1 ACTGTACCACAGATCTG ATAGCAGTGGCTGGTAC TGGG ------TACTTCCAGCA 3-66 2 D2-2 0 JH3TACTGTGCGAG--- TC GTAGTACCAGCTGCTAT GATGCTTTTGATGTCT 3-21 6 D2-21 8 JH4TTACTGTGCGAG--- TTTTGG GTGGTGGT CACATTTA --------GACTACTGGGG 4-39 8D3-10 3 JH4 ACTGTGCGAGACA TGAAAAAC TTCGGGGAGTTAT AAT---------CTACTGGGGCC 1-69 7 D6-13 1 JH4 TTACTGTGCGAG--- GGGGATGGCAGCAGCTGGTAC C -------GACTACTGGGGC 1-8 0 D2-2 12 JH2 ACTGTGCGAGAG-ATTGTAGTAGTACCAGCTGC CAAGATCGTAAG ----TGGTACTTCGAT 1-18 0 D5-24 15 JH4TTACTGTGC------ GAGATGG GTTTTTGATCCCCAG -----TTTGACTACTGG 3-20 4 D7-27 1JH3 TCACTGTGCGAGAA TTTT ACTGGGGA T GATGCTTTTGATGTCT

HUMAN λ NUCLEOTIDE SEQUENCES Vλ N Jλ 1-17 AGCCTGAGTGGTC-- 2 (TT) Jλ3--------TTCGGCGGAGGG 2-13 CAGTGGTAACCATCT 0 Jλ2 ---GGTATTCGGCGGAGG 1-19CAGCCTGAGTGCTG- 0 Jλ1 -----TCTTCGGAACTGGG 5-2 AGCAACTTCGTGTA- 2 (TA) Jλ3------GTTCGGCGGAGAG 1-7 GGTAGTAGCACTT-- 1 (C) J3 --------TCGGCGGAGGGA2-13 CAGTGGTAACCAT-- 0 Jλ1 -TATGTCTTCGGAACTG 2-1 GACAGCAGCACT--- 0 Jλ1-TATGTCTTCGGAACTG 1-2 GGCAGCAACAATTTC 1 (G) Jλ1 --ATGTCTTCGGAACTG 1-4AGCAGCAGCACTC-- 2 (GT) Jλ3 -------TTCGGCGGAGG 1-4 AGCAGCAGCACTC--- 0 Jλ1----------GGAACTGGGA

Characterization of Human Antibody Produced in HAC Calves

HAC transgenic calves produced as described above were examined fortheir production of human antibody using a solid phase ELISA assay(FIGS. 41-44). Among forty-two calves examined, all forty-two calves hadan antibody titer that was higher than background. The highest human Iglevel shown in Table 4 is 10000 ng/ml. Because Ig levels fluctuate, Iglevels tested on other days or later in development may yield muchhigher values. Seven calves had a level of at least 2000 ng/ml. Theseresults demonstrate that fully human antibodies can be purified from theserum of HAC transchromosomal cattle (FIG. 42). Furthermore, thepresence of mu and gamma heavy chains demonstrates that the HACundergoes class switching at the human heavy chain locus within atranschromosomal ungulate (e.g., a bovine). The HAC-encoded human lightchains (lambda) are bound to human mu chains and to human gamma chains(FIG. 43). This result demonstrates that the xenogeneic B cells ofHAC-transchromosomal animals are capable of assembling human heavy andlight chains.

Adjuvant-stimulated immunization of HAC-transchromosomal cattlegenerates a polyclonal, antigen-specific response to the immunizingantigen (FIG. 41). The reaction of the antibody to different epitopes ofthe immunizing antigen demonstrates that HAC transchromosomal animalscan recognize multiple epitopes of a complex antigen. Human mu, gamma,and light chains were found in human Ig purified from aHAC-transchromosomal calf in which an antigen-specific human antibodyresponse was induced (FIG. 44). TABLE 4 Human Ig Levels in HACTransgenic Animals ANIMAL HUMAN IG IDENTIFICATION LEVEL IN Number NUMBERng/ml 1 100 10000 2 104 4000 3 1098 4000 4 1075 3000 5 1163 3000 6 822556 7 1076 2000 8 68 497 9 1098 403 10 1064 258 11 1093 253 12 1065 21013 71 160 14 80 141 15 1075 134 16 1076 100 17 73 98 18 72 93 19 1066 7020 67 69 21 50 55 22 86 48 23 1094 33 24 77 30 25 89 30 26 88 30 27 107729 28 74 25 29 1098 25 30 1079 24 31 91 22 32 1081 21 33 76 20 34 66 1735 90 17 36 1076 16 37 1088 15 38 87 15 39 95 15 40 1092 13 41 1068 1342 1090 12

Table 4 contains measurements of the human Ig levels (ng/ml) produced byHAC animals. The measurements were performed using a solid phase ELISAassay with a highly specific polyclonal bovine anti-human antibody as acapture reagent and polyclonal bovine anti-human antibody conjugated tobiotin as the detection reagent.

EXAMPLE 2 Evidence for Nuclear Reprogramming Deficiencies in TraditionalBovine Nuclear Transplant Embryos

Traditional nuclear transplant techniques generally produce a lowpercentage of live births. As described below, this in efficiency may bedue to, at least in part, the inability of the reconstituted oocyte toreprogram the donor cell or donor nucleus to promote the transcriptionof genes desirable for development of the oocyte and to inhibit thetranscription of genes undesirable for development. Examples belowdescribe improved cloning methods that can be used to produce transgenicungulates expressing xenogenous antibodies in the methods of the presentinvention.

Distribution of Nuclear Envelope, Nuclear Matrix and Chromatin-MatrixInterface Components during Bovine Preimplantation Development Todetermine the distribution of nuclear envelope (B-type and A/C-typelamins), nuclear matrix (NuMA), and chromatin-matrix interface (AKAP95)components in preimplantation embryos, bovine embryos were produced byin vitro fertilization (IVF) and examined by immunofluorescenceanalysis. Bovine in vitro fertilization was performed as describedpreviously (Collas et al., Mol. Reprod. Devel. 34:212-223, 1993).Briefly, frozen-thawed bovine sperm from a single bull was layered ontop of a 45-90% Percoll gradient and centrifuged for 30 minutes at700×g. The concentration of sperm in the pellet was determined, and thesperm was diluted such that the final concentration at fertilization was10⁶ sperm/ml. At 22 hours post maturation, oocytes were washed threetimes in TL HEPES and placed in 480 μl fertilization medium. Twenty μlsperm suspension were added at 10⁶ sperm/ml for 50 oocytes. Embryos wereplaced in culture in four-well tissue culture plates containing amonolayer of mouse fetal fibroblasts in 0.5 ml of embryo culture mediumcovered with 0.3 ml of embryo tested mineral oil (Sigma). Between 25 and50 embryos were placed in each well and incubated at 38.5° C. in a 5%CO₂ air atmosphere. Fertilization rates were over 90% as determined bypronuclear development.

For the immunofluorescence analysis of these in vitro fertilized bovineembryos, anti-human lamin B antibodies were obtained from Dr.Jean-Claude Courvalin, CNRS, Paris, France. Anti-lamins A/C monoclonalantibodies were purchased from Santa-Cruz Biotechnology, and anti-NuMAantibodies were obtained from Transduction Laboratories. Anti-rat AKAP95affinity-purified rabbit polyclonal antibodies were obtained fromUpstate Biotechnologies. The in vitro fertilized bovine embryos weresettled onto poly-L-lysine-coated glass coverslips, fixed with 3%paraformaldehyde for 15 minutes, and permeabilized with 0.1% TritonX-100 for 15 minutes (Collas et al., J. Cell Biol. 135:1715-1725, 1996).The proteins were blocked with 2% BSA in PBS/0.01% Tween 20 (PBST) for15 minutes. Primary antibodies (anti-AKAP95, anti-lamin B, anti-LBR,anti-NuMA, and anti-lamins A/C) and secondary antibodies were incubatedeach for 30 minutes and used at a 1:100 dilution in PBST-BSA. DNA wascounterstained with 0.1 μg/ml Hoechst 33342 incorporated in the antifademounting medium. Samples were mounted onto slides and coverslips sealedwith nail polish. Immunofluorescence observations were made on anOlympus BX60 epifluorescence microscope and photographs were taken witha JVC CCD camera and AnalySIS software. Images were processed using theAldus Photostyler software. Relative quantification of fluorescencesignals was performed using the AnalySIS quantification program. Datawere expressed as mean relative fluorescence intensities.

Immunofluorescence analysis of bovine embryos showed that B-type laminswere detected at the nuclear periphery (FIG. 29A). Lamins A/C, however,were not detected at the pronuclear or 8-cell stage. This failure todetect lamins A/C at these early cell stages is expected for a marker ofdifferentiated cells (Guilli et al., EMBO J. 6:3795-3799, 1987). Thenuclear matrix structural protein, NuMA, was detected in all the stagesthat were examined (FIG. 29A). However, in bovine pronuclear stageembryos, NuMA labeling was restricted to the female pronucleus (FPN),the smallest of both pronuclei (FIG. 29A arrows). AKAP95, which wasrecently characterized in early mouse embryos (Bomar et al., 2002manuscript submitted) and detected using affinity-purified anti-ratAKAP95 antibodies, was also restricted to the female pronucleus (FIG.29A). Nevertheless, intranuclear distribution of AKAP95 was observed innuclei of all blastomeres in subsequent developmental stages (FIG. 29A).

Specificity of immunofluorescence labeling was verified by Western blotanalysis of bovine primary fetal fibroblasts and pronuclear stage invitro fertilized embryos (FIG. 29B). For this analysis, proteins wereresolved by 10% SDS-PAGE at 40 mA per gel. Proteins wereelectrophoretically transferred onto a nitrocellulose membrane intransfer buffer (25 mM Tris HCl, pH 8.3, 192 mM glycine, 20% methanol,and 0.1% SDS) at 100 V for one hour. Membranes were washed for 10minutes with Tris-buffered saline (TBS; i.e., 140 mM NaCl, 2.7 mM KCl,and 25 mM Tris-HCl at pH 8.0), blocked for one hour with TBST (TBS with0.05% Tween-20) containing 5% milk, and incubated for 1.5 hours with thefollowing primary antibodies: anti-AKAP95 (1:250 dilution), anti-lamin B(1:1000), anti-LBR (1:500), anti-NuMA (1:500), and anti-lamins A/C(1:500). Blots were washed twice for 10 minutes in TBST and incubatedfor one hour with horse radish peroxidase (HRP)-conjugated secondaryantibodies. Blots were washed twice for 10 minutes in TBS and developedusing enhanced chemiluminescence (ECL, Amersham).

All proteins were detected at their expected apparent M_(r): 68 kDa(B-type lamins), 70 and 60 kDa (lamins A and C, respectively), ˜180 kDa(NuMA), and 95 kDa (AKAP95). Altogether, these results indicate thatpreimplantation bovine embryos express nuclear structural proteins thatcan be detected with cross-reacting antibodies. Notably, lamins A/C arenot immunologically detected in bovine preimplantation embryos. Becauselamins A/C are expressed in somatic cells (FIG. 29B), they potentiallyconstitute molecular markers for nuclear reprogramming in nucleartransplant embryos.

Dynamics of Nuclear Envelope, Numa, and AKAP95 in Nuclear TransplantBovine Embryos The dynamics of nuclear envelope and nuclear matrixstructures was examined during traditional nuclear transplantationprocedure in bovine. These structures were investigated using antibodiesto lamins A/C and B, NuMA, and AKAP95, respectively. To determine thedynamics of these markers during nuclear remodeling, bovine nucleartransplant embryos were produced using primary fetal fibroblasts, whichwere isolated as described previously, as the donor cells (Kasinathan etal., Biol. Reprod. 64:1487-1493, 2001). Briefly, cells were harvestedfrom bovine fetuses by trypsinization using 0.08% trypsin and 0.02% EDTAin PBS (trypsin-EDTA). Cells were seeded in a T75 culture flask(Corning) in α-MEM (Gibco) supplemented with 10% fetal bovine serum(FBS; Hyclone), 0.15 g/ml glutamine (Sigma), 0.003% β-mercaptoethanol(Gibco), and an antibiotic-antimycotic (Gibco). On day three afterseeding, cells were harvested with trypsin-EDTA and frozen inα-MEM/DMSO. G1 cells were isolated as described previously (Kasinathanet al., Biol. Reprod. 64:1487-1493, 2001). Briefly, 24 hours beforeisolation, 5.0×10⁵ cells were plated in a T75 flask containing 10 ml ofMEM/FBS. The following day, the plates were washed with PBS, the culturemedium was replaced for 1-2 hours, and the plates were shaken for 30-60seconds on a Vortex at medium speed. The medium was removed, centrifugedat 500×g for five minutes, and the pellet was resuspended in 250 μl ofMEM/FBS. Cell doublets attached by a cytoplasmic bridge were selectedusing a micropipette and used for nuclear transfer.

Bovine nuclear transfer was carried out as described earlier (Kasinathanet al., Biol. Reprod. 64:1487-1493, 2001). In vitro-matured oocytes wereenucleated 18-20 hours post-maturation. After transferring G1 donorcells into the perivitelline space, they were fused using a singleelectrical pulse of 2.4 kV/cm for 20 microseconds (ElectrocellManipulator 200, Genetronics). At 28-30 hours post maturation (i.e.,28-30 hours after oocytes were placed in maturation medium aftercollection from ovaries and at least two hours after fusion with donorcells) reconstructed oocytes and parthenogenetic controls were activatedwith calcium ionophore (5 μM) for four minutes (Cal Biochem) followed by10 μg cycloheximide and 2.5 μg cytochalasin D (Sigma) in ACM medium (100mM NaCl, 3 mM KCl, 0.27 mM CaCl₂, 25 mM NaHCO₃, 1 mM sodium lactate, 0.4mM pyruvate, 1 mM L-glutamine, 3 mg/ml BSA, 1% BME amino acids, and 1%MEM nonessential amino acids, for five hours (Liu et al., Mol. Reprod.Dev. 49:298-307, 1998). After activation, nuclear transplant embryos oroocytes eggs were washed five times and co-cultured with mouse fetalfibroblasts at 38.5° C. in a 5% CO₂ atmosphere.

Reconstituted embryos were activated using standard methods, and threehours post-fusion, embryos at the premature chromatin condensation (PCC)stage were fixed with paraformaldehyde and analyzed byimmunofluorescence using antibodies to lamins A/C, lamin B, NuMA, andAKAP95 (FIG. 30, PCC). Furthermore, groups of nuclear transplant embryosthat were allowed to progress to the pronuclear (PN) stage (i.e., 15hour post-fusion bovine embryos) were analyzed similarly (FIG. 30,nuclear transplant-PN). As controls, parthenogenetic oocytes activatedas described herein were also examined at the pronuclear stage (FIG. 30,Parth. PN).

As expected, somatic donor cells (bovine fetal fibroblasts, FIG. 30)expressed all markers with a distribution anticipated from theliterature. At the premature chromatin condensation stage, distinctcondensed chromosome masses were evidenced by DNA staining with Hoechst33342. Lamins A/C and B were not detected on or near the condensedchromosomes (FIG. 30, PCC), presumably as a result of their dispersal inthe egg cytoplasm. Some labeled NuMA was detected; this NuMA waspresumably associated with the spindle poles maintaining the condensedchromosomes. AKAP95, in contrast, was associated with the condensed(PCC) chromosomes. This result is reminiscent of AKAP95 labeling inmitotic human cells (Collas et al., J. Cell Biol. 147:1167-1180, 1999;Steen et al., J. Cell Biol. 150:1251-1262, 2000). At the pronuclearstage, all markers were detected. Lamins A/C were present at thepronuclear envelope (FIG. 2, nuclear transplant-PN). This contrastedwith their absence from the envelope of control parthenote pronuclei(FIG. 30) and from the envelope of fertilized pronuclei (FIG. 29A).Lamin B was detected in nuclear transplant pronuclei, as in controlpronuclei. Likewise, NuMA and AKAP95 decorated the nuclear interiorexcept for the nucleoli. NuMA labeling was consistently brighter innuclear transplant pronuclei than in control parthenogenetic pronuclei(compare nuclear transplant PN and Parth. PN, FIG. 30). Collectively,these observations indicate that pronuclei of nuclear transplant embryosreassemble the somatic nuclear markers lamins A and C and display strongNuMA staining.

Differential Anchoring of AKAP95 in Pronuclei of Parthenogenetic Embryosand Nuclear Transplant Embryos The A-kinase anchoring protein AKAP95 isa nuclear protein implicated in mitotic chromosome condensation. For useas another molecular marker affecting reprogramming of somatic nucleiafter nuclear transplant, the intranuclear anchoring properties ofAKAP95 were characterized in bovine nuclear transplant pronuclear stageembryos formed from fetal fibroblasts. Anchoring of AKAP95 in pronucleifrom parthenogenetic embryos and nuclei of somatic donor cells was alsoexamined.

Intranuclear anchoring of AKAP95 in pronuclear embryos was examined insitu by extraction of embryos with 0.1% Triton X-100, 1 mg/ml DNAse 1,and either 100 or 300 mM NaCl for 30 minutes at room temperature. Asnoted above, male pronuclei did not harbor any AKAP95. In contrast, asignificant amount of AKAP95 and DNA was resistant to DNAse 1 and 300 mMNaCl in pronuclei of nuclear transplant embryos, and in donor nuclei inbovine (FIG. 31). B-type lamins were not extracted by DNAse 1 and 300 mMNaCl in parthenote or nuclear transplant pronuclei (FIG. 31), suggestingthat alterations in AKAP95 and DNA distributions did not result fromgross changes in nuclear architecture. These data indicate that, as insomatic nuclei, AKAP95 is more tightly anchored to intranuclearstructures in nuclear transplant pronuclei than in parthenogeneticpronuclei in the bovine. Whether this association imposes constraints onDNA organization or results from altered genome organization in nucleartransplant embryos remains to be determined. As DNAse I-resistant DNA istranscriptionally silent, incomplete remodeling of AKAP95 anchoringafter nuclear transplantation likely impairs expression ofdevelopmentally important genes.

Transcriptional Misregulation of Lamins A/C in Nuclear Transplant BovineEmbryos A striking observation was that lamins A/C reassemble at theperiphery of pronuclei in bovine nuclear transplant embryos, whereasthis somatic-specific marker is absent from in vitro fertilized, andparthenogenetic pronuclei. Thus, we investigated whether reassembly oflamins A/C resulted from (i) re-targeting of somatic lamins disassembledat the premature chromatin condensation stage (FIG. 30), (ii)translation and assembly of lamins from a pool of maternal lamin A/CmRNA, or (iii) de novo transcription of the somatic lamin A (LMNA) genein nuclear transplant pronuclei.

To distinguish between these possibilities, bovine nuclear transplantembryos were produced by either the “traditional” nuclear transplantprocedure as described herein, nuclear transplant followed by activationof reconstituted embryos with the protein synthesis inhibitorcycloheximide (CHX), or by nuclear transplant followed by activation inthe presence of the RNA polymerase II (PolII) inhibitor actinomycin D(ActD) to inhibit de novo transcription. For culturing bovine nucleartransplant embryos in cycloheximide, oocytes were activated afternuclear transfer as described above except that oocytes were incubatedfor 14 hours in cycloheximide (CHX). At 14 hours after activation,oocytes were washed five times and placed in ACM culture mediumcontaining 15 μg/ml Hoechst 33342 (Sigma) for one hour. Afterincubation, pronuclear development was observed by epifluorescencemicroscopy. Pronuclear embryos were then fixed in 3% paraformaldehyde inPBS, washed, and mounted on slides. For culturing bovine nucleartransplant oocytes in actinomycin D, oocytes were activated afternuclear transfer as described above except 5 μg/ml actinomycin D (ActD)was added to the cycloheximide incubation step. After five hours, eggswere washed five times and placed in ACM culture medium containing 5μg/ml actinomycin D. At 14 hours after activation, eggs were washed fivetimes and placed in ACM culture medium containing 15 μg/ml Hoechst 33342(Sigma) for one hour. After incubation, pronuclear development wasobserved by epifluorescence microscopy. Pronuclear stage embryos werefixed in 3% paraformaldehyde in PBS, washed, and mounted on slides.

Lamin B assembly around nuclear transplant pronuclei was not affected byeither protein or RNA synthesis inhibition. This result indicates thatlamin B was reassembled from either a previously disassembled somaticpool and/or from a large pool of lamin B in the oocyte cytoplasm. LaminsA/C, which were detected in nuclear transplant pronuclei (FIG. 30), wereabsent from nuclei reformed after activation with cycloheximide. Thisresult indicates that lamins A/C assembly requires de novo proteinsynthesis and that these lamins are not re-targeted from a disassembledsomatic pool brought into the oocyte by donor nucleus injection or cellfusion. Furthermore, lamins A/C are not reassembled when embryos areactivated in the presence of actinomycin D. This result indicates thatlamins A/C reassembly in nuclear transplant pronuclei results from denovo transcription of the LMNA gene in the reconstituted pronucleus.NuMA, which was detected in nuclear transplant pronuclei, is notreassembled in pronuclei of nuclear transplant embryos activated withcycloheximide, but is faintly detected in pronuclei of actinomycinD-treated nuclear transplant embryos. This finding strongly suggeststhat NuMA reassembly in nuclear transplant pronuclei requires de novotranslation that occurs, at least in part, from a pool of maternal NuMAmRNA. The consistent observation that anti-NuMA labeling is weaker inpronuclei of actinomycin D-treated nuclear transplant embryos comparedto control untreated nuclear transplant embryos (compare b′ and b′″ inFIG. 32) suggests that part of NuMA assembly in nuclear transplantpronuclei results from de novo transcription of the NuMA gene at thepronuclear stage.

Collectively, these results indicate that the LMNA gene is not turnedoff upon nuclear remodeling after nuclear transplantation. Similarly,the NuMA gene apparently remains active in pronuclear nuclear transplantembryos. It is likely that transient inactivation of these genes takesplace during premature chromatin condensation, as anticipated from thehighly condensed nature of the chromatin (FIG. 30). These resultsclearly illustrate incomplete nuclear reprogramming in nucleartransplant embryos produced under the conditions described herein. Asdiscussed earlier for AKAP95, we propose that the persistence of laminsA/C in nuclear transplant pronuclei affects gene expression, such asexpression of developmentally important genes. The previously reportedinteractions of lamins A and C with chromatin proteins and DNA, and theassociation of these lamins with transcription factors also support thishypothesis.

EXAMPLE 3 Exemplary Nuclear Reprogramming Deficiencies in TraditionalBovine Nuclear Transplant Embryos

Exemplary differences between naturally-occurring embryos andtraditional nuclear transfer embryos are also described below. Thesedifferences include differences in pronuclear assembly of differentiatedcell-specific A-type nuclear lamins, enhanced pronuclear NuMA and TATAbinding protein concentrations, and increased sensitivity of nuclearmatrix-chromatin interface component AKAP95 and DNA to extraction withdetergent, DNAse, and salt.

For these studies, bovine fetal fibroblast cell lines were establishedas described previously (Kasinathan et al., Nat. Biotechnol.19:1176-1178, 2001 and Kasinathan et al., Biol. Reprod. 64:1487-1493,2001). G1-phase fibroblast doublets were isolated from cultures using apreviously described shake-off method (Kasinathan et al., Nat.Biotechnol. 19:1176-1178, 2001). In vitro fertilization with invitro-matured oocytes was carried out as described previously (Collas etal., Mol. Reprod. Dev. 34:224-231, 1993). For nuclear transplantation(NT) and oocyte activation, nuclear transplantation using G1-phase donorcells was performed at ˜20 hours post-maturation (hpm) as reportedpreviously (Kasinathan et al., Nat. Biotechnol. 19:1176-1178, 2001 andKasinathan et al., Biol. Reprod. 64:1487-1493, 2001). Reconstitutedembryos were activated at 28-30 hpm (T=0) with 5 μM calcium ionophorefor four minutes followed by 10 μg/ml CHX and 2.5 μg/ml cytochalasin Dfor five hours. Embryos were washed and co-cultured with mouse fetalfibroblasts (Kasinathan et al., Biol. Reprod. 64:1487-1493, 2001). Whenreconstituted embryos were cultured in CHX, oocytes were activated asabove and cultured with 2.5 μg/ml CHX for another nine hours (total, 14hours in CHX). Embryos were washed thoroughly and cultured as described(Kasinathan et al., Biol. Reprod. 64:1487-1493, 2001). Whenreconstituted embryos were exposed to ActD, oocytes were activated asabove except that 5 μg/ml ActD was added to the five hour CHX incubationstep.

For immunological analysis, cells, oocytes, embryos, nuclei, andchromatin masses were settled onto poly-L-lysine-coated coverslips,fixed with 3% paraformaldehyde for 15 minutes, and permeabilized with0.1% Triton X-100 for 15 minutes. Proteins were blocked using PBS/2%BSA/0.01% Tween 20. Primary and secondary antibodies (1:100 dilution)were incubated each for 30 minutes. In particular, rabbit polyclonalantibodies against a peptide of human lamin B were used (Chaudhary etal., J. Cell Biol. 122:295-306, 1993). Goat anti-lamin B polyclonalantibodies, anti-lamin A/C monoclonal antibodies, and anti-TBPantibodies from Santa-Cruz Biotechnology were also used. Anti-NuMAmonoclonal antibodies were from Transduction Laboratories, and anti-ratAKAP95 affinity-purified polyclonal antibodies were from UpstateBiotechnologies. DNA was counterstained with 0.1 μg/ml Hoechst 33342.Photographs were taken with a JVC CCD camera, and quantification ofimmunofluorescence intensity was performed using the AnalySIS software.Data were expressed as a mean ±SD fluorescence intensity relative to acontrol in at least three replicates. For in situ extractions, embryosand cells settled on coverslips were incubated for 15 minutes with 0.1%Triton X-100, 1 mg/ml DNAse I, and 300 mM NaCl in Tris-HCl (pH 7.2)prior to immunofluorescence analysis. For immunoblotting, 100 embryoswere dissolved in 20 μl SDS sample buffer, proteins resolved by 10%SDS-PAGE, and analyzed with the following antibodies: anti-lamin B,1:1,000; anti-lamin A/C, 1:250; anti-NuMA, 1:500; anti-AKAP95, 1:250.

Based on the above immunological analysis, the distribution of A/C- andB-type lamins, NuMA and AKAP95 was characterized in bovine fetalfibroblasts commonly used for NT. In in vitro-produced bovinepreimplantation embryos, lamin B was detected at the nuclear peripheryas early as the pronuclear (PN) stage. Lamin A/C was absent, as expectedfrom a marker of differentiated cells. NuMA and AKAP95 were restrictedto the female pronucleus at the pronuclear stage but decorated allnuclei in subsequent stages. Specificity of immunofluorescence data wasverified on immunoblots.

The dynamics of lamins A/C and B, NuMA, and AKAP95 was examined duringmorphological nuclear remodeling associated with transplantation ofbovine fibroblasts into enucleated oocytes by electrofusion (Kasinathanet al. Biol. Reprod. 64:1487-1493, 2001). Donor nuclei underwentpremature chromatin condensation (PCC) within three hours of fusion.Nuclear lamins and NuMA were redistributed in the oocyte cytoplasm andwere absent from PCC chromosomes. AKAP95 was associated with PCCchromosomes, a property reminiscent of mitotic cells. Fourteen hoursafter start of activation treatment of recipient oocytes, NT embryosdisplayed fully developed pronuclei. However, in contrast to pronucleiof parthenogenetic or fertilized embryos, essentially all NT pronucleiexpressed strong lamin A/C and NuMA immunoreactivity, twocharacteristics of the somatic donor cells.

Recipient oocyte activation in the presence of 10 μg/ml of the proteinsynthesis inhibitor cycloheximide (CHX) or 5 μg/ml of the RNA polymerase(Pol) II inhibitor actinomycin D (ActD), both compatible with pronuclearformation, inhibited pronuclear lamin A/C assembly. This resultindicates that assembly of these somatic lamins results fromtranscription of the somatic lamin A (LAMA) gene. Lamin B assembly wasnot perturbed by CHX or ActD, indicating that it was re-targeted from adisassembled somatic pool and/or from a maternal pool of B-type lamins.Essentially no NuMA was detected after CHX exposure; however, 40% ofNuMA immunoreactivity in NT pronuclei was detected after ActD treatment.This result suggests that NuMA assembles as a result of translation frommaternal mRNA and of de novo transcription. As lamin A/C and NuMA areabnormally transcribed in NT pronuclei, these proteins can be used asmarkers to determine the ability of nuclear transfer methods toreprogram the donor genetic material.

As discussed above, the intranuclear anchoring properties of AKAP95, astructural multivalent protein of the nuclear matrix-chromatin interface(Collas et al., J. Cell Biol. 147:1167-1179, 1999) and enriched inhypoacetylated chromatin, can also be used as a marker for reprogrammingof donor genetic material. AKAP95 was the only marker investigated thatwas detected in somatic donor nuclei on PCC chromosomes and in NTpronuclei with a labeling intensity similar to that of parthenotes andPN embryos. AKAP95 association with NT pronuclei was maintained byinhibition of protein or RNA synthesis. Thus, a major fraction of PNAKAP95 in NT embryos is of somatic origin. AKAP95 anchoring was examinedby extraction of NT embryos, parthenotes, and donor fibroblasts with0.1% Triton X-100, 1 mg/ml DNAse 1 and 300 mM NaCl. In parthenotes, 90%of AKAP95 and DNA were extracted; however, 35% of AKAP95 in NT pronucleiresisted extraction. Sensitivity of AKAP95 and DNA to DNAse I and NaClresembled that of fibroblast nuclei. Lamin B was not extracted underthese conditions, indicating that differences in AKAP95 and DNAextractability did not result from gross alterations in nucleararchitecture. These results imply that NT pronuclei are characterized bytight anchoring of AKAP95 and restricted DNA accessibility to DNAse I.Thus, pronuclei produced by somatic NT appear to display structuralabnormalities as a result of incomplete morphological remodeling ofdonor nuclei and/or transcriptional misregulation of somatic genes.

EXAMPLE 4 Additional Exemplary Nuclear Reprogramming Deficiencies inTraditional Bovine Nuclear Transplant Embryos

As described above, expression patterns in Nuclear Transplant (NT)embryos were compared to those in in vitro-produced (IVP)preimplantation embryos. For this comparison, in vitro fertilization wasperformed, and embryos were cultured as previously described (Collas etal., Mol. Reprod. Dev. 34:224-231, 1993 and Kasinathan et al., Biol.Reprod. 64:1487-1493, 2001). NT was carried out by fusing donor bovinefetal fibroblasts to enucleated oocytes (Kasinathan et al., Nat.Biotechnol. 19:1176-1178, 2001 and Kasinathan et al., Biol. Reprod.64:1487-1493, 2001). Recipient oocytes were activated at 28-30 hourspost-maturation (hpm) with 5 μM calcium ionophore for 4 minutes followedby 10 μg/ml CHX and 2.5 μg/ml cytochalasin D for 5 hours and washed.Embryos were co-cultured with mouse fetal fibroblasts (Kasinathan etal., Biol. Reprod. 64:1487-1493, 2001). For CHX treatment, oocytes wereactivated as above and embryos were cultured with 2.5 μg/ml CHX foranother nine hours before culture. For ActD treatment, oocytes wereactivated as above except that 5 μg/ml ActD was added to the five-hourCHX incubation step and embryos were maintained in 5 μg/ml ActD foranother nine hours prior to culture. NT embryos were cultured to theblastocyst stage in vitro, and two embryos were transferred perrecipient female. Pregnancies were monitored by ultrasonography, andC-sections were performed. Calves were scored by veterinarians within 24hours of birth.

For analysis of protein levels in NT and IVP embryos, anti-lamin Bpolyclonal antibodies, anti-lamin A/C monoclonal antibodies, andanti-TBP polyclonal or monoclonal antibodies from Santa-CruzBiotechnology were used. Anti-AKAP95 antibodies were from UpstateBiotechnologies. Immunofluorescence analysis was performed as described(Bomar et al., J. Cell Sci. 115:2931-2940, 2002). Briefly, cells andembryos were settled onto poly-L-lysine-coated coverslips, fixed with 3%paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100for 15 minutes, and proteins were blocked in PBS/2% BSA/0.01% Tween 20.Samples were incubated with primary and secondary antibodies (1:100dilutions) each for 30 minutes. DNA was counterstained with 0.1 μg/mlHoechst 33342. Photographs were taken with a JVC CCD camera, andquantification of immunofluorescence intensity performed using theAnalySIS software. When indicated, samples were extracted on coverslipswith a cocktail of 1% Triton X-100, 1 mg/ml DNAse I, and 300 mM NaCl inTris-HCl (pH 7.2) for 15 minutes prior to immunofluorescence analysis.For immunoblotting, protein samples (30 μg) were resolved by 10%SDS-PAGE, blotted onto nitrocellulose, and probed with indicatedantibodies.

Dynamics of the donor nucleus in nuclear transplant embryos Toinvestigate the dynamics of somatic nuclei during bovine nucleartransplantation (NT), the distribution of two structural components ofthe nuclear envelope, the ubiquitously expressed B-type lamins (referredto as lamin B) and the differentiated cell-specific A-type lamins (laminA/C) was examined (Gruenbaum et al., J. Struct. Biol. 129:313-323,2000). Nuclear lamins anchor nuclear membranes to chromatin and havebeen suggested to promote nuclear expansion after (pro)nuclearreconstitution in vitro. Lamins have also been shown to be essential forcell survival, as failure to assemble B-type lamins leads to cell death.As a marker of the transcription machinery, the dynamics of theTATA-binding protein, TBP, a transcription factor for virtually allgenes, was analyzed. Perinuclear distribution of lamin B andcolocalization of TBP with DNA in bovine fetal fibroblasts and in invitro-produced (IVP) preimplantation embryos were consistent withobservations in other species (Holy et al., Dev. Biol. 168:464-478,1995; Houliston et al., Development 102:271-278, 1988; and Worrad etal., Development 120:2347-2357, 1994). Lamin A/C was not detected duringpreimplantation development as expected from a marker of differentiatedcells. Specificity of immunofluorescence labeling was verified onimmunoblots.

Following transplantation of fibroblast nuclei into enucleated oocytes,both lamins A/C and B were disassembled from the prematurely condensedchromosomes, while TBP remained associated with the chromosomes.Fourteen hours after initiation of activation of the recipient oocytes,all NT embryos contained fully developed pronuclei with perinuclearlamin B labeling and TBP co-localized with DNA. However, in contrast toIVP embryos, 95-99% of NT embryos displayed lamin A/C expression asearly as the pronuclear stage, and expression persisted during earlydevelopment (see below). Relative amounts of immunolabeled lamin B,lamin A/C, and TBP in pronuclei of NT and IVP embryos were quantified bymeasuring the ratio of secondary antibody fluorescence intensity to thatof DNA (Hoechst 33342) to account for DNA content (haploid vs diploid)in the nuclei examined. Whereas relative amounts of lamin B were similarin pronuclei of NT and IVP embryos, relative mounts of lamin A/C and TBPwere higher in NT pronuclei than in male (MPN) or female (FPN) pronucleiin IVP embryos.

TBP, DNA, and A-type lamins in pronuclei of NT embryos Higher amounts ofTBP in NT pronuclei was associated with a greater resistance to in situextraction with a combination of detergent (1% Triton X-100), nuclease(1 mg/ml DNAse 1), and salt (0.3 M NaCl). Quantification ofimmunofluorescence labeling intensity in extracted embryos relative tothat of non-extracted controls shows that ˜35% of TBP remainedunextracted in male (MPN) or female (FPN) pronuclei of IVP embryos.However, TBP of NT pronuclei displayed strong resistance to extractionas in fibroblast nuclei. Similarly, DNA of NT pronuclei displayed a4.5-fold increase in resistance to extraction under these conditionscompared to pronuclei of IVP embryos, suggestive of a more compactchromatin organization.

To determine the origin of lamin B, lamin A/C, and TBP in pronuclei ofNT embryos, recipient oocytes were activated in the presence of the RNApolymerase (Pol) II inhibitor actinomycin D (ActD; 5 μg/ml) or with theprotein synthesis inhibitor cycloheximide (CHX; 10 μg/ml) (Knott et al.,Biol. Reprod. 66:1095-1103, 2002) as described herein. Assembly of laminA/C, lamin B, and TBP was examined by densitometric analysis ofimmunofluorescently labeled pronuclear embryos. Both inhibitorsprevented pronuclear lamin A/C assembly, suggesting that assembly ofthese somatic lamins in NT embryos resulted from transcription of thelamin A gene at the pronuclear stage. Lamin B assembly was not perturbedby CHX or ActD treatment, suggesting that lamin B was assembled fromsomatic lamins solubilized in the oocyte cytoplasm after NT and/or froma maternal pool of lamins. Similar amounts of TBP were detected inuntreated embryos or after inhibition of RNA or protein synthesis. AsTBP associates with condensed chromosomes during PCC, and since themetaphase II oocyte cytoplasm is devoid of detectable TBP, TBP ofsomatic origin probably remains associated with the donor genome duringNT. Thus, in addition to expressing A-type lamins, pronuclei of NTembryos display higher amounts and enhanced intranuclear anchoring ofTBP.

EXAMPLE 5 Use of Reprogrammed Donor Chromatin Masses to Clone Mammals

To overcome the problem of incomplete reprogramming in traditionalnuclear transfer embryos that was demonstrated above, new methods weredeveloped to more efficiently reprogram donor chromatin prior to nucleartransfer (PCT/US01/50406, filed Dec. 21, 2001). These methods involveincubating a nucleus (e.g., a nucleus that encodes a xenogenousantibody) from a donor cell in a reprogramming media (e.g., a cellextract) that results in nuclear envelope dissolution and possiblychromatin condensation. This nuclear envelope breakdown and chromatincondensation allows the release of transcription regulatory proteinsthat were attached to the chromosomes and that would otherwise promotethe transcription of genes undesirable for oocyte, embryo, or fetusdevelopment. Additionally, regulatory proteins from the reprogrammingmedia may bind the chromatin mass and promote the transcription of genesdesirable for development.

To generate an ungulate expressing a xenogenous antibody, the donornucleus or chromatin mass can be modified before, during, or afterreprogramming by insertion of one or more nucleic acids encoding axenogenous antibody. If desired, a cell from the cloned fetus or thecloned offspring can be used in a second round of nuclear transfer togenerate additional cloned offspring. Cells from the initial clonedfetus or cloned offspring may also be frozen to form a cell line to beused as a source of donor cells for the generation of additional clonedungulates.

Bulk Preparation of Donor Nuclei for Use in Cloning As many as severalmillion nuclei may be isolated from synchronized or unsynchronized cellpopulations in culture. The cell populations may be synchronizednaturally or chemically. Preferably, at least 40, 60, 80, 90, or 100% ofthe cells in a population are arrested in G_(o) or G₁ phase. Toaccomplish this, cells may be incubated, for example, in low serum, suchas 5%, 2%, or 0% serum, for 1, 2, 3, or more days to increase thepercentage of cells in G_(o) phase. To synchronize cells in G₁, thecells may be grown to confluence as attached cells and then incubated in0.5-1 μg/ml nocodazole (Sigma Chemicals, St. Louis, Mo.) for 17-20hours, as described previously (see, for example, Collas et al., J. CellBiol. 147:1167-1180, 1999 and references therein). The flasks containingthe attached cells are shaken vigorously by repeatedly tapping theflasks with one hand, resulting in the detachment of mitotic cells andG₁ phase doublets. The G₁ phase doublets are pairs of elongated cells atthe end of the division process that are still connected by a thinbridge. Detached G₁ phase doublets may be isolated from the media basedon this characteristic doublet structure. The G₁ phase doublets mayremain attached or may divide into two separate cells after isolation.

The synchronized or unsynchronized cells are harvested in phosphatebuffered saline (PBS) using standard procedures, and several washingsteps are performed to transfer the cells from their original media intoa hypotonic buffer (10 mM HEPES, pH 7.5, 2 mM MgCl₂, 25 mM KCl, 1 mMDTT, 10 μM aprotinin, 10 μM leupeptin, 10 μM pepstatin A, 10 μM soybeantrypsin inhibitor, and 100 μM PMSF). For example, the cells may bewashed with 50 ml of PBS and pelleted by centrifugation at 500×g for 10minutes at 4° C. The PBS supernatant is decanted, and the pelleted cellsare resuspended in 50 ml of PBS and centrifuged, as described above.After this centrifugation, the pelleted cells are resuspended in 20-50volumes of ice-cold hypotonic buffer and centrifuged at 500×g for 10 minat 4° C. The supernatant is again discarded and approximately 20 volumesof hypotonic buffer are added to the cell pellet. The cells arecarefully resuspended in this buffer and incubated on ice for at leastone hour, resulting in the gradual swelling of the cells.

To allow isolation of the nuclei from the cells, the cells are lysedusing standard procedures. For example, 2-5 ml of the cell suspensionmay be transferred to a glass homogenizer and Dounce homogenized usingan initial 10-20 strokes of a tight-fitting pestle. Alternatively, thecell suspension is homogenized using a motorized mixer (e.g.,Ultraturrax). If desired, cell lysis may be monitored using phasecontrast microscopy at 40-fold magnification. During thishomogenization, the nuclei should remain intact and most or preferablyall of the originally attached cytoplasmic components such as vesicles,organelles, and proteins should be released from the nuclei. Ifnecessary, 1-20 μg/ml of the cytoskeletal inhibitors, cytochalasin B orcytochalasin D, may be added to the aforementioned hypotonic buffer tofacilitate this process. Homogenization is continued as long asnecessary to lyse the cells and release cytoplasmic components from thenuclei. For some cell types, as many as 100, 150, or more strokes may berequired. The lysate is then transferred into a 15 ml conical tube onice, and the cell lysis procedure is repeated with the remainder of thesuspension of swollen cells. Sucrose from a 2 M stock solution made inhypotonic buffer is added to the cell lysate (e.g., ⅛ volume of 2 Mstock solution is added to the lysate), resulting in a finalconcentration of 250 mM sucrose. This solution is mixed by inversion,and the nuclei are pelleted by centrifugation at 400×g in a swing outrotor for 10 to 40 minutes at 4° C. The supernatant is then discarded,and the pelleted nuclei are resuspended in 10-20 volumes of nuclearbuffer (10 mM HEPES, pH 7.5, 2 mM MgCl₂, 250 mM sucrose, 25 mM KCl, 1 mMDTT, 10 μM aprotinin, 10 μM leupeptin, 10 μM pepstatin A, 10 μM soybeantrypsin inhibitor, and 100 μM PMSF). The nuclei are sedimented andresuspended in 1-2 volumes of nuclear buffer, as described above. Thefreshly isolated nuclei may either be used immediately for in vitroreprogramming and nuclear transfer as described below or stored forlater use. For storage, the nuclei are diluted in nuclear buffer to aconcentration of approximately 10⁶/ml. Glycerol (2.4 volumes of 100%glycerol) is added and mixed well by gentle pipetting. The suspension isaliquoted into 100-500 μl volumes in 1.5-ml tubes on ice, immediatelyfrozen in a methanol-dry ice bath, and stored at −80° C. Prior to use,aliquots of the nuclei are thawed on ice or at room temperature. Onevolume of ice cold nuclear buffer is added, and the solution iscentrifuged at 1,000×g for 15 minutes in a swing out rotor. The pelletednuclei are resuspended in 100-500 μl nuclear buffer and centrifuged asdescribed above. The pelleted nuclei are then resuspended in a minimalvolume of nuclear buffer and stored on ice until use.

Preparation of Mitotic Extract or Media for Use in Reprogramming DonorGenetic Material For the preparation of a mitotic extract, a somaticcell line (e.g., fibroblasts) is synchronized in mitosis by incubationin 0.5-1 μg/ml nocodazole for 17-20 hours (e.g., Collas et al., J. CellBiol. 147:1167-1180, 1999 and references therein) and the mitotic cellsare detached by vigorous shaking, as described above. The detached G₁phase doublets may be discarded, or they may be allowed to remain withthe mitotic cells which constitute the majority off the detached cells(typically at least 80%). The harvested detached cells are centrifugedat 500×g for 10 minutes in a 10 ml conical tube at 4° C. Several cellpellets are pooled, resuspended in a total volume of 50 ml of cold PBS,and centrifuged at 500×g for 10 minutes at 4° C. This PBS washing stepis repeated. The cell pellet is resuspended in approximately 20 volumesof ice-cold cell lysis buffer (20 mM HEPES, pH 8.2, 5 mM MgCl₂, 10 mMEDTA, 1 mM DTT, 10 μM aprotinin, 10 μM leupeptin, 10 μM pepstatin A, 10μM soybean trypsin inhibitor, 100 μM PMSF, and optionally 20 μg/mlcytochalasin B), and the cells are sedimented by centrifugation at 800×gfor 10 minutes at 4° C. The supernatant is discarded, and the cellpellet is carefully resuspended in no more than one volume of cell lysisbuffer. The cells are incubated on ice for one hour to allow swelling ofthe cells. The cells are lysed by either sonication using a tipsonicator or Dounce homogenization using a glass mortar and pestle. Celllysis is performed until at least 90% of the cells and nuclei are lysed,which may be assessed using phase contrast microscopy. The sonicationtime required to lyse at least 90% of the cells and nuclei may varydepending on the type of cell used to prepare the extract.

The cell lysate is placed in a 1.5-ml centrifuge tube and centrifuged at10,000 to 15,000×g for 15 minutes at 4° C. using a table top centrifuge.The tubes are removed from the centrifuge and immediately placed on ice.The supernatant is carefully collected using a 200 μl pipette tip, andthe supernatant from several tubes is pooled and placed on ice. Thissupernatant is the “mitotic cytoplasmic” or “MS15” extract. This cellextract may be aliquoted into 50 μl or 10 μl volumes of extract per tubeon ice, depending on whether the regular or micromethod for generationof chromatin masses will be used. The extracts are immediatelyflash-frozen on liquid nitrogen and stored at −80° C. until use.Alternatively, the cell extract is placed in an ultracentrifuge tube onice (e.g., fitted for an SW55 Ti rotor; Beckman). If necessary, the tubeis overlayed with mineral oil to the top. The extract is centrifuged at200,000×g for three hours at 4° C. to sediment membrane vesiclescontained in the MS15 extract. At the end of centrifugation, the oil isdiscarded. The supernatant is carefully collected, pooled if necessary,and placed in a cold 1.5 ml tube on ice. This supernatant is referred toas “MS200” or “mitotic cytosolic” extract. The extract is aliquoted andfrozen as described for the MS15 extract.

If desired, the extract can be enriched with additional nuclear factors.For example, nuclei can be purified from cells of the cell type fromwhich the reprogramming extract is derived or from cells of any othercell type and lysed by sonication as described above. The nuclearfactors are extracted by a 10-60 minute incubation in nuclear buffercontaining NaCl or KCl at a concentration of 0.15-800 mM underagitation. The lysate is centrifuged to sediment unextractablecomponents. The supernatant containing the extracted factors of interestis dialyzed to eliminate the NaCl or KCl. The dialyzed nuclear extractis aliquoted and stored frozen. This nuclear extract is added at variousconcentrations to the whole cell extract described above prior to addingthe nuclei for reprogramming.

Mitotic extracts can also be prepared from germ cells, such as oocytesor male germ cells. For example, metaphase II oocytes that are naturallyarrested at this stage can be harvested, washed, and lysed as describedabove for the generation of an oocyte extract. To prepare a male germcell extract, germ cells are isolated from testes obtained from theabattoir by mincing the organ and by differential centrifugation of theharvested cells on a sucrose or percoll gradient. Germ cells areseparated from somatic (Leydig and Sertoli) cells, washed by suspension,and sedimentation in PBS. The cells are then washed once in ice-soldcell lysis buffer as described above and lysed by sonication. The lysateis cleared by centrifugation at 15,000×g for 15 minutes at 4° C., andthe supernatant (i.e., the germ cell extract) is aliquoted andsnap-frozen in liquid nitrogen.

As an alternative to a cell extract, a reprogramming media can also beformed by adding one or more naturally-occurring or recombinant factors(e.g., nucleic acids or proteins such as DNA methyltransferases, histonedeacetylases, histones, protamines, nuclear lamins, transcriptionfactors, activators, repressors, chromatin remodeling proteins, growthfactors, interleukins, cytokines, or other hormones) to a solution, suchas a buffer. Preferably, one or more of the factors are specific foroocytes or stem cells.

Formation of Condensed Chromatin Masses by Exposure of Nuclei to aMitotic Extract or Media An aliquot of MS15 or MS200 extract or themitotic media is thawed on ice. An ATP-generating system (0.6 μl) isadded to 20 μl of extract or media and mixed by vortexing. For thepreparation of the ATP-generating system, equal proportions of 100 mMATP stock, 1 M creatine phosphate, and 2.5 mg/ml creatine kinase stocksolutions (100×) made in H₂O are mixed and stored on ice until use.After addition of the ATP generating system to the extract, the finalconcentrations are 1 mM ATP, 10 mM creatine phosphate, and 25 μg/mlcreatine kinase.

The nuclei suspension is added to the extract or media at aconcentration of 1 μl nuclei per 10 μl of extract or media, mixed wellby pipetting, and incubated in a 30, 33, 35, 37, or 39° C. water bath.The tube containing the mixture is tapped gently at regular intervals toprevent chromosomes from clumping at the bottom of the tube. Nuclearenvelope breakdown and chromosome condensation is monitored at regularintervals, such as every 15 minutes, under a microscope. When thenuclear envelope has broken down and chromosomes have started tocondense, the procedure for recovery of chromatin masses from theextract or media is started.

Formation of Decondensed Chromatin Masses by Exposure of Nuclei to aMitotic Extract or Media and Anti-Numa Antibodies Alternatively,chromatin masses that are not condensed or only partially condensed maybe formed by performing the above procedure after pre-loading theisolated nuclei with an antibody to the nuclear matrix protein NuMA(Steen et al., J. Cell Biol. 149, 531-536, 2000). This procedure allowsthe removal of nuclear components from chromatin by the dissolution ofthe nuclear membrane surrounding the donor nuclei; however, thecondensation step is inhibited by addition of the anti-NuMA antibody.Preventing chromosome condensation may reduce the risk of chromosomebreakage or loss while the chromosomes are incubated in the mitoticextract.

For this procedure, purified cell nuclei (2,000 nuclei/μl) arepermeabilized in 500 μl nuclear buffer containing 0.75 μg/mllysolecithin for 15 minutes at room temperature. Excess lysolecithin isquenched by adding 1 ml of 3% BSA made in nuclear buffer and incubatingfor 5 minutes on ice. The nuclei are then sedimented and washed once innuclear buffer. The nuclei are resuspended at 2,000 nuclei/μl in 100 μlnuclear buffer containing an anti-NuMA antibody (1:40 dilution;Transduction Laboratories). After a one hour incubation on ice withgentle agitation, the nuclei are sedimented at 500×g through 1 M sucrosefor 20 minutes. The nuclei are then resuspended in nuclear buffer andadded to a mitotic extract or media containing an ATP regeneratingsystem, as described in the previous section. Optionally, the anti-NuMAantibody may be added to the extract or media to further preventchromosome condensation.

Formation of Decondensed Chromatin Masses by Exposure of Nuclei to aDetergent and/or Salt Solution or to A Protein Kinase Solution Chromatinmasses that are not condensed or only partially condensed may also beformed by exposure to a detergent or protein kinase. Detergent may beused to solubilize nuclear components that are either unbound or looselybound to the chromosomes in the nucleus, resulting in the removal of thenuclear envelope. For this procedure, purified cell nuclei (2,000-10,000nuclei/μl) are incubated in nuclear buffer supplemented with adetergent, such as 0.1% to 0.5% Triton X-100 or NP-40. To facilitateremoval of the nuclear envelope, additional salt, such as NaCl, may beadded to the buffer at a concentration of approximately 0.1, 0.15, 0.25,0.5, 0.75, or 1 M. After a 30-60 minute incubation on ice with gentleshaking, the nuclei are sedimented by centrifugation at 1,000×g in aswing-out rotor for 10-30 minutes, depending on the total volume. Thepelleted nuclei are resuspended in 0.5 to 1 ml nuclear buffer andsedimented as described above. This washing procedure is repeated twiceto ensure complete removal of the detergent and extra salt.

Alternatively, the nuclear envelope may be removed using recombinant ornaturally-occurring protein kinases, alone or in combination.Preferably, the protein kinases are purified using standard proceduresor obtained in purified form from commercial sources. These kinases mayphosphorylate components of the nuclear membrane, nuclear matrix, orchromatin, resulting in removal of the nuclear envelope (see, forexample, Collas and Courvalin, Trends Cell Biol. 10: 5-8, 2000).Preferred kinases include cyclin-dependent kinase 1 (CDK1), proteinkinase C (PKC), protein kinase A (PKA), MAP kinase,calcium/calmodulin-dependent kinase (CamKII), and CK1 casein kinase, orCK2 casein kinase. For this method, approximately 20,000 purified nucleiare incubated in 20 μl of phosphorylation buffer at room temperature ina 1.5 ml centrifuge tube. A preferred phosphorylation buffer for CDK1(Upstate Biotechnology) contains 200 mM NaCl, 50 mM Tris-HCl (pH7.2-7.6), 10 mM MgSO₄, 80 mM β-glycerophosphate, 5 mM EGTA, 100 μM ATP,and 1 mM DTT. For PKC, a preferred buffer contains 200 mM NaCl, 50 mMTris-HCl (pH 7.2-7.6), 10 mM MgSO₄, 100 μM CaCl₂, 40 μg/mlphosphatidylserine, 20 μM diacylglycerol, 100 μM ATP, and 1 mM DTT. Ifboth PKC and CDK1 are used simultaneously, the CDK1 phosphorylationbuffer supplemented with 40 μg/ml phosphatidylserine and 20 μMdiacylglycerol is used. A preferred phosphorylation buffer for PKAincludes 200 mM NaCl, 10 mM MgSO4, 10 mM Tris, pH 7.0, 1 mM EDTA, and100 μM ATP. For MAP kinase, the PKA phosphorylation buffer supplementedwith 10 mM CaCl₂, and 1 mM DTT may be used. For CamKII, either PKAbuffer supplemented with 1 mM DTT or a Cam Kinase assay kit from UpstateBiotechnology (Venema et al. J. Biol. Chem. 272: 28187-90, 1997) isused.

The phosphorylation reaction is initiated by adding a protein kinase toa final amount of 25-100 ng. The reaction is incubated at roomtemperature for up to one hour. Nuclear envelope breakdown may bemonitored by microscopy during this incubation, such as at 15 minuteintervals. After nuclear envelope breakdown, nuclei are washed threetimes, as described above for the removal of the detergent solution.

Recovery of Chromatin Masses from the Media, Extract, Detergent and/orSalt Solution, or Protein Kinase Solution The extract or solutioncontaining the condensed, partially condensed, or not condensedchromatin masses is placed under an equal volume of 1 M sucrose solutionmade in nuclear buffer. The chromatin masses are sedimented bycentrifugation at 1,000×g for 10-30 minutes depending on the samplevolume in a swing out rotor at 4° C. The supernatant is discarded, andthe pelleted chromatin masses are carefully resuspended by pipetting in0.1-1.0 ml nuclear buffer or lipofusion buffer (150 mM NaCl, 10 μMaprotinin, 10 μM leupeptin, 10 μM pepstatin A, 10 μM soybean trypsininhibitor, and 100 μM PMSF in either 20 mM HEPES around pH 7.0 or pH 7.5or 20 mM MES around pH 6.2) and centrifuged at 1,000×g for 10-30minutes. The supernatant is discarded, and the pelleted chromatin massesare resuspended in nuclear buffer or lipofusion buffer and stored on iceuntil use. Each chromatin mass is transferred to a 20 μl drop ofHEPES-buffered medium under oil in a micromanipulation dish. Onechromatin mass is inserted into each enucleated oocyte, as describedbelow.

Micromethod for Preparation of Chromatin Masses A 10-20 μl drop of MS15or MS200 extract or mitotic media containing an ATP generating system, adetergent and/or salt solution, or a protein kinase solution asdescribed above is placed in a petri dish. A 50-μl drop of isolated G₁phase cell doublets or G₀ phase cells in culture medium, a separate 50μl “lysis” drop of HEPES- or bicarbonate-buffered medium containing 0.1%Triton X-100 or NP-40 for use in facilitating cell lysis, and a 50-μldrop of oocyte injection medium is then added. Each of these drops iscovered with CO₂ equilibrated mineral oil. A 50 μl “wash drop” ofculture medium is also added to the petri dish for use in washing thelysed cells or nuclei.

Cells are transferred to the lysis drop using a micropipette. The cellmembranes are lysed in the pipette by gentle repeated aspirations. Whenthe cell is lysed, the lysate is gently expelled into the wash drop, andthe nucleus is immediately reaspirated to remove detergent. Optionally,the nuclei may be permeabilized and incubated with anti-NuMA antibodiesprior to being added to the mitotic extract or media. The nucleus isthen expelled into the drop of MS15, MS200, or media, detergent and/orsalt solution, or protein kinase solution. Nuclear breakdown andchromosome condensation is monitored as described above. Once thenuclear envelope has broken down and, if a mitotic extract withoutanti-NuMA antibodies was used, the chromosomes have started to condense,a single intact chromatin mass is isolated with a micropipette andtransferred to an enucleated recipient oocyte, as described below.

Enucleation of Oocytes Preferably, the recipient oocyte is a metaphaseII stage oocyte. At this stage, the oocyte may be activated or isalready sufficiently activated to treat the introduced chromatin mass asit does a fertilizing sperm. For enucleatation of the oocyte, part orpreferably all of the DNA in the oocyte is removed or inactivated. Thisdestruction or removal of the DNA in the recipient oocyte prevents thegenetic material of the oocyte from contributing to the growth anddevelopment of the cloned mammal. One method for destroying thepronucleus of the oocyte is exposure to ultraviolet light (Gurdon, inMethods in Cell Biology, Xenopus Laevis:—Practical Uses in cell andMolecular Biology, Kay and Peng, eds., Academic Press, California,volume 36:pages 299-309, 1991). Alternatively, the oocyte pronucleus maybe surgically removed by any standard technique (see, for example,McGrath and Solter, Science 220:1300-1319, 1983). In one possiblemethod, a needle is placed into the oocyte, and the nucleus is aspiratedinto the inner space of the needle. The needle may then be removed fromthe oocyte without rupturing the plasma membrane (U.S. Pat. Nos.4,994,384 and 5,057,420).

Lipofusion for Insertion of Chromatin Masses into Oocytes Chromatin maybe introduced into recipient oocytes by lipofusion as described below orby standard microinjection or electrofusion techniques (see, forexample, U.S. Pat. Nos. 4,994,384 and 5,945,577). The followinglipofusion method may also be used in other applications to insertchromosomes into other recipient cells.

Chromatin masses are isolated from the mitotic extract, detergent and/orsalt solution, or protein kinase solution by centrifugation, and thenwashed with lipofusion buffer, as described above. The chromatin massesmay be in stored in ice-cold lipofusion buffer until use. Alternatively,the chromatin masses are aliquoted, frozen in liquid nitrogen or in amethanol-dry ice bath, and stored frozen at −80° C. The lipofusionsolution is prepared by mixing one or more fusogenic reagents with thelipofusion buffer in respective proportions ranging from 5:1 to 1:10approximately. The fusogenic reagents consist of, but are not limitedto, polyethylene glycol (PEG) and lipophilic compounds such asLipofectin®, Lipofectamin®, DOTAP®{N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylamonium methylsulfate;C₄₃H₈₃NO₈S}, DOSPA®{2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate}, and DOPE® (dioleoyl phosphatidylethanolamine).

Other preferred lipids include neutral and monovalent or multivalentcationic lipids, such as those containing quaternary ammonium groups.Additional preferred lipids have a cholesterol moiety such as thatformed from the reaction of the hydroxyl group in cholesterol with agroup in the lipid. Still other preferred lipids have a saturated orunsaturated fatty acid that preferably contains between 5 and 10, 10 and15, 15 and 20, or 20 and 30 carbon atoms, inclusive. These lipids may besynthesized using standard chemical synthesis techniques, obtained fromnaturally-occurring sources, or purchased from commercially availablesource (Summers et al., Biophys J. 71(6):3199-206, 1996; Nabekura etal., Pharm Res. 13(7): 1069-72, 1996; Walter et al., Biophys J. 66(2 Pt1):366-376, 1994; Yang et al., Biosci Rep. 13(3):143-157, 1993; Walterand Siegel, Biochemistry. 6:32(13):3271-3281, 1993). Other preferredfusogenic compounds are phospholipids such as membrane vesicle fractionsfrom sea urchin eggs or any other source (Collas and Poccia, J. of CellScience 109, 1275:1283, 1996). Preferably, contacting chromosomes withthe membrane vesicle fraction does not result in the chromosomes beingencapsulated by an intact membrane.

For example, a cationic lipid, such as DOTAP®, may be used at aconcentration of approximately 0.1 to 30 μg/ml in lipofusion buffer.Alternatively, a liposome formulation consisting of a mixture of acationic lipid and a neutral lipid, such as DOPE®, may be used.

The chromatin masses, either freshly prepared or frozen and thawed, aremixed with the lipofusion solution to allow coating of the chromatinmasses with the compound. Incubation takes place at a temperature of20-30° C. for a period of approximately 10-30 minutes. Microdropscontaining the chromatin masses in the lipofusion solution are placedunder CO₂ equilibrated mineral oil. A drop containing the enucleatedrecipient oocytes is also prepared. The chromatin masses coated with thelipofusion reagent are picked up in a micropipette and inserted in theperivitellin space, between the oocyte cytoplasm and the zona pellucida.The chromatin mass is placed next to the oocyte membrane to ensurecontact with the oocyte. The chromatin mass-oocyte complexes aremaintained at a temperature of 20-30° C., and fusion is monitored underthe microscope. Once fusion has occurred, reconstituted oocytes areactivated as described below.

Activation Culturing and Transplantation of Reconstituted Oocytes Toprevent polar body extrusion and chromosome loss, the oocyte may beactivated in the presence of cytochalasin B, or cytochalasin B may beadded immediately after activation (Wakayama et al., PNAS96:14984-14989, 1999; Wakayama et al., Nature Genetics 24:108-109,2000). Either electrical or non-electrical means may be used foractivating reconstituted oocytes. Electrical techniques for activatingcells are well known in the art (see, for example, U.S. Pat. Nos.4,994,384 and 5,057,420). Non-electrical means for activating cells mayinclude any method known in the art that increases the probability ofcell division. Examples of non-electrical means for activating an oocyteinclude incubating the oocyte in the presence of ethanol; inositoltrisphosphate; Ca⁺⁺ ionophore and a protein kinase inhibitors; a proteinsynthesis inhibitor; phorbol esters; thapsigargin, or any component ofsperm. Other non-electrical methods for activation include subjectingthe oocyte to cold shock or mechanical stress. Alternatively, one tothree hours after nuclear transfer, oocytes may be incubated forapproximately six hours in medium containing Sr²⁺ to activate them andcytochalasin B to prevent cytokinesis and polar body extrusion (Wakayamaet al., PNAS 96:14984-14989, 1999; Wakayama et al., Nature Genetics24:108-109, 2000). Depending on the type of mammal cloned, the preferredlength of activation may vary. For example, in domestic animals such ascattle, the oocyte activation period generally ranges from about 16-52hours or preferably about 28-42 hours.

After activation, the oocyte is placed in culture medium for anappropriate amount of time to allow development of the resulting embryo.At the two cell stage or a later stage, the embryo is transferred into afoster recipient female for development to term. For bovine species, theembryos are typically cultured to the blastocyst stage (e.g., forapproximately 6-8 days) before being transferred to maternal hosts. Forother cloned animals, an appropriate length for in vitro culturing isknown by one skilled in the art or may be determined by routineexperimentation.

Methods for implanting embryos into the uterus of a mammal are also wellknown in the art. Preferably, the developmental stage of the embryo iscorrelated with the estrus cycle of the host mammal. Once the embryo isplaced in the uterus of the mammal, the embryo may develop to term.Alternatively, the embryo is allowed to develop in the uterus until achosen time, and then the embryo (or fetus) is removed using standardsurgical methods to determine its health and viability. Embryos from onespecies may be placed into the uterine environment of an animal fromanother species. For example, bovine embryos can develop in the oviductsof sheep (Stice and Keefer, Biology of Reproduction 48: 715-719, 1993).Any cross-species relationship between embryo and uterus may be used inthe methods of the invention.

Lipofusion of Nuclei with Oocytes or Other Recipient Cells Thelipofusion solution is prepared by mixing one or more fusogenic reagentswith lipofusion buffer in respective proportions ranging fromapproximately 5:1 to 1:10, as described above. Nuclei, either freshlyprepared or frozen and thawed as described above, are mixed with thelipofusion solution to allow coating of the nuclei with the compound.Incubation takes place at a temperature of 20-30° C. for a period ofapproximately 10-30 minutes. Microdrops containing nuclei in thelipofusion solution are placed under CO₂ equilibrated mineral oil. Adrop containing the recipient cell, preferably an enucleated cell, isalso prepared. Enucleated recipient cells are prepared by physicallyremoving the chromosomes or the nucleus by micromanipulation or bydamaging the genetic material by exposure to UV light, as describedabove. For insertion into oocytes, the nuclei coated with the lipofusionreagent are picked up in a micropipette and inserted in the perivitellinspace, between the oocyte cytoplasm and the zona pellucida. Forinsertion into other recipient cells, the coated nuclei are preferablyplaced next to the cell membrane to ensure contact with the cell. Thenucleus-cell complexes are maintained at a temperature of 20-30° C., andfusion is monitored using a microscope. Once fusion has occurred,reconstituted oocytes are activated as described above.

EXAMPLE 6 Use of Reprogrammed Permeabilized Cells to Clone Mammals

Cells may also be reprogrammed without requiring the isolation of nucleior chromatin masses from the cells. In this method, cells arepermeabilized and then incubated in an interphase or mitoticreprogramming media under conditions that allow the exchange of factorsbetween the media (e.g., a cell extract) and the cells. If an interphasemedia is used, the nuclei in the cells remain membrane-bounded; if amitotic media is used, nuclear envelope breakdown and chromatincondensation may occur. After the nuclei are reprogrammed by incubationin this media, the plasma membrane is preferably resealed, forming anintact reprogrammed cell that contains desired factors from the media.If desired, the media can be enriched with additional nuclear factors asdescribed herein. The reprogrammed cells are then fused with recipientoocytes, and embryos formed from the reconstituted oocytes are insertedinto maternal recipient mammals for the generation of cloned mammals.For the production of an ungulate expressing a xenogenous antibody, thedonor cells are modified before, during, or after programming by theinsertion of a nucleic acid encoding a xenogenous antibody. If desired,a cell from the cloned fetus or the cloned offspring can be used in asecond round of nuclear transfer to generate additional clonedoffspring. Cells from the initial cloned fetus or cloned offspring mayalso be frozen to form a cell line to be used as a source of donor cellsfor the generation of additional cloned ungulates.

Permeabilization of Cells Cells that may be reprogrammed using thisprocedure include unsynchronized cells and cells synchronized in G_(o),G₁, S, G₂, or M phase or a combination of these phases. The cells arepermeabilized using any standard procedure, such as permeabilizationwith digitonin or Streptolysin O. Briefly, cells are harvested usingstandard procedures and washed with PBS. For digitonin permeabilization,cells are resuspended in culture medium containing digitonin at aconcentration of approximately 0.001-0.1% and incubated on ice for 10minutes. For permeabilization with Streptolysin O, cells are incubatedin Streptolysin O solution (see, for example, Maghazachi et al., FASEBJ. 11:765-74, 1997, and references therein) for ˜15, 30, or 60 minutesat room temperature. After either incubation, the cells are washed bycentrifugation at 400×g for 10 minutes. This washing step is repeatedtwice by resuspension and sedimentation in PBS. Cells are kept in PBS atroom temperature until use. Preferably, the permeabilized cells areimmediately added to the interphase or mitotic media for reprogramming,as described below.

Preparation of the Reprogramming Media To Prepare an Interphasereprogramming extract, interphase cultured cells are harvested usingstandard methods and washed by centrifugation at 500×g for 10 minutes ina 10 ml conical tube at 4° C. The supernatant is discarded, and the cellpellet is resuspended in a total volume of 50 ml of cold PBS. The cellsare centrifuged at 500×g for 10 minutes at 4° C. This washing step isrepeated, and the cell pellet is resuspended in approximately 20 volumesof ice-cold interphase cell lysis buffer (20 mM HEPES, pH 8.2, 5 mMMgCl₂, 1 mM DTT, 10 μM aprotinin, 10 μM leupeptin, 10 μM pepstatin A, 10μM soybean trypsin inhibitor, 100 μM PMSF, and optionally 20 μg/mlcytochalasin B). The cells are sedimented by centrifugation at 800×g for10 minutes at 4° C. The supernatant is discarded, and the cell pellet iscarefully resuspended in no more than one volume of interphase celllysis buffer. The cells are incubated on ice for one hour to allowswelling of the cells. The cells are lysed by either sonication using atip sonicator or Dounce homogenization using a glass mortar and pestle.Cell lysis is performed until at least 90% of the cells and nuclei arelysed, which may be assessed using phase contrast microscopy. Thesonication time required to lyse at least 90% of the cells and nucleimay vary depending on the type of cell used to prepare the extract.

The cell lysate is placed in a 1.5-ml centrifuge tube and centrifuged at10,000 to 15,000×g for 15 minutes at 4° C. using a table top centrifuge.The tubes are removed from the centrifuge and immediately placed on ice.The supernatant is carefully collected using a 200 μl pipette tip, andthe supernatant from several tubes is pooled and placed on ice. Thissupernatant is the “interphase cytoplasmic” or “IS15” extract. This cellextract may be aliquoted into 20 μl volumes of extract per tube on iceand immediately flash-frozen on liquid nitrogen and stored at −80° C.until use. Alternatively, the cell extract is placed in anultracentrifuge tube on ice (e.g., fitted for an SW55 Ti rotor;Beckman). If necessary, the tube is overlayed with mineral oil to thetop. The extract is centrifuged at 200,000×g for three hours at 4° C. tosediment membrane vesicles contained in the IS15 extract. At the end ofcentrifugation, the oil is discarded. The supernatant is carefullycollected, pooled if necessary, and placed in a cold 1.5 ml tube on ice.This supernatant is referred to as “IS200” or “interphase cytosolic”extract. The extract is aliquoted and frozen as described for the IS15extract.

If desired, the extract can be enriched with additional nuclear factors.For example, nuclei can be purified from cells of the cell type fromwhich the reprogramming extract is derived or from cells of any othercell type and lysed by sonication as described above. The nuclearfactors are extracted by a 10-60 minute incubation in nuclear buffercontaining NaCl or KCl at a concentration of 0.15-800 mM underagitation. The lysate is centrifuged to sediment unextractablecomponents. The supernatant containing the extracted factors of interestis dialyzed to eliminate the NaCl or KCl. The dialyzed nuclear extractis aliquoted and stored frozen. This nuclear extract is added at variousconcentrations to the whole cell extract described above prior to addingthe cells for reprogramming.

Interphase extracts can also be prepared from germ cells, such asoocytes or male germ cells. For example, oocytes are activated asdescribed above and cultured for five hours to allow entry intointerphase. Oocytes are then treated as described herein for metaphaseII oocyte extracts except that EDTA is omitted from the lysis buffer.Male germ cell extracts can be prepared as described herein.

As an alternative to a cell extract, a reprogramming media can also beformed by adding one or more naturally-occurring or recombinant factors(e.g., nucleic acids or proteins such as DNA methyltransferases, histonedeacetylases, histones, protamines, nuclear lamins, transcriptionfactors, activators, repressors, chromatin remodeling proteins, growthfactors, interleukins, cytokines, or other hormones) to a solution, suchas a buffer. Preferably, one or more of the factors are specific foroocytes or stem cells.

Reprogramming of Cells in a Media The permeabilized cells are suspendedin an interphase reprogramming media described above or one of themitotic reprogramming medias described herein at a concentration ofapproximately 100-1,000 cells/μl. The ATP generating system and GTP areadded to the extract as described above, and the reaction is incubatedat 30-37° C. for up to two hours to promote translocation of factorsfrom the extract into the cell and active nuclear uptake orchromosome-binding of factors. The reprogrammed cells are centrifuged at800×g, washed by resuspension, and centrifuged at 400×g in PBS. Ifdesired, the cells are resuspended in culture medium containing 20-30%fetal calf serum (FCS), RPMI1640 containing 2 mM CaCl₂ (added from a 1 Mstock in H₂O), or in α-MEM medium containing 2 mM CaCl₂ and incubatedfor 1-3 hours at 37° C. in a regular cell culture incubator to allowresealing of the cell membrane. The cells are then washed in regularwarm culture medium (10% FCS) and cultured further using standardculturing conditions. Alternatively, the reprogrammed permeabilizedcells may be used for genetic transfer to oocytes without resealing thecell membrane.

Alternative Method of Reprogramming Permeabilized Cells on CoverslipsInstead of in Solution Alternatively, the cells can be permeabilizedwhile placed on coverslips to minimize the handling of the cells and toeliminate the centrifugation of the cells, thereby maximizing theviability of the cells. Cells (e.g., fibroblasts) are grown on 16-mmpoly-L-lysine-coated coverslips in RPMI1640 to 50,000-100,000cells/coverslip in 12-well plates. Cells are permeabilized in 200 ng/mlStreptolysin O in Ca²⁺-free Hanks Balanced Salt Solution (Gibco-BRL) for50 minutes at 37° C. in regular atmosphere. If desired, the percent ofcells that are permeabilized under these conditions can be measuredbased on propidium iodide uptake. Streptolysin O is aspirated;coverslips are overlaid with 80-100 μl of reprogramming media; and thecells are incubated for thirty minutes to one hour at 37° C. in CO₂atmosphere. The reprogramming media preferably contains the ATPgenerating system and 1 mM each of ATP, CTP, GTP and UTP. To optionallyreseal plasma membranes, α-MEM medium containing 2 mM CaCl₂, mediumcontaining 20-30% fetal calf serum, or RPMI1640 containing 2 mM CaCl₂ isadded to the wells, and the cells are incubated for two hours at 37° C.Alternatively, the plasma membrane is not resealed.

Effect of Various Streptolysin O Treatments on the Percentage ofPermeabilized and Resealed Cells To assess the percent of permeabilizedand resealed cells, dose and time titrations of Streptolysin Oincubation were performed (Table 5). Permeabilization of cells wasassessed by uptake of 0.1 μg/ml of the DNA stain propidium iodide at theend of Streptolysin O treatment. Resealing was assessed similarly at theend of the resealing treatment in a separate group of cells. TABLE 5Permeabilization and resealing of Streptolysin O (SLO)-treated bovinefibroblasts Permeabilization Resealing ng/ml SLO N % pemeabilized +/− sdN % Resealed +/− sd 0 563   1 +/− 2.8 560 89.9 +/− 4.9 100 404 48.6 +/−4.2 810 86.1 +/− 8.3 200 548 79.2 +/− 1.4 478 84.9 +/− 1.5 500 495 88.7+/− 1.6 526 87.6 +/− 0.5 1000 425 84.9 +/− 0.7 544 86.4 +/− 1.4 2000 31596.6 +/− 2.2 425 10.7 +/− 1   4000 200   99 +/− 1.4 200 11.2 +/− 5.3

Assessment of Viability of Bovine Fibroblasts Permeabilized withStreptolysin O Treatment and Exposed to Mitotic Extract TUNEL analysiswas performed to evaluate apoptosis in cells permeabilized with 0 or 500ng/ml Streptolysin O and resealed, or in cells permeabilized withStreptolysin O, exposed to mitotic extract for 30 or 60 minutes, andresealed. TUNEL-positive cells are cells undergoing apoptosis (i.e.,cell death). The data show that Streptolysin O itself does not induceapoptosis (Table 6). Exposure of Streptolysin O-treated cells to themitotic extract for 60 minutes, but not 30 minutes, induces a 10%increase in apoptotic rate, based on TUNEL analysis (Table 6). Based onthese data, a 30-minute incubation of donor cells in the extract is morepreferable than a 60 minute incubation. Thirty minute incubations wereshown by immunofluorescence analysis of cells to induce nuclear envelopebreakdown in the majority of nuclei examined (˜90%, n>100).

Additionally, purified nuclei incubated in extract and washed in eitherbuffer N or TL-HEPES and sucrose as described herein for the chromatintransfer method do not undergo apoptosis (2/34 and 3/47 TUNEL positive,respectively). TABLE 6 TUNEL analysis of Streptolysin O and StreptolysinO plus extract-treated bovine fibroblasts ng/ml SLO N % TUNEL pos. +/−sd 0-Input cells 400 7.7 +/− 1.7  0 800  6.5 +/− 0.17 500 892  7.3 +/−3.41  0 + extract 30′ 400  5.5 +/− 1.12 500 + extract 30′ 400 8.2 +/−1.1  0 + extract 60′ 784 6.5 +/− 4.0 500 + extract 60′ 691 16.9 +/− 1.9 The permeabilization method chosen for these cloning methods was 500ng/ml SLO for 30 minutes at 38° C. The resealing method chosen forforming an intact membrane surrounding the reprogrammed cells was a twohour incubation in α-MEM medium containing 2 mM CaCl₂.

Alternatively Cell Permeabilized Method using a Protease such as TrypsinIn an exemplary cell permeabilization method using a protease, cells(e.g., fibroblasts) are grown to confluency in a 35 mm plate overnight.The fibroblasts are removed from the plate using the normal trypsin-EDTA(0.5%-5.3 mM) procedure for five minutes. The fibroblasts are washed inPBS and then resuspended in 1 ml TL Hepes. One ml of 3 mg/ml protease inTL Hepes is added to 1 ml of cell suspension (final proteaseconcentration is 1.5 mg/ml) and incubated at room temperature for oneminute. The cells are washed in 10 ml HBSS and resuspended in 1 ml HBSSand counted. Approximately 10⁵ cells in minimal volume are used formitotic extract incubation. Forty ul of MS-15 mitotic extract is placedon the cells for 30 minutes at 37° C. Cells are then washed in TL Hepesand used in one of the nuclear transfer procedures described herein.

These cells show signs of permeabilization because after incubation inthe mitotic extract, 70-80% of the cells have undergone nuclear envelopebreakdown and premature chromosome condensation, indicating that MPF(maturation promoting factor) from the extract or some component of MPFhas traversed the membrane. This cell permeabilization method can beused with a variety of proteases such as trypsin and with a variety ofgerm, somatic, embryonic, fetal, adult, differentiated, andundifferentiated cells.

Formation Activation, Culturing, and Transplantation of ReconstitutedOocytes The reprogrammed cells are inserted into, or fused with,recipient oocytes using standard microinjection or electrofusiontechniques (see, for example, U.S. Pat. Nos. 4,994,384 and 5,945,577).For example, the cells can be placed next to the oocytes in standardcell medium in the presence or absence of sucrose (e.g., 2.5% sucrose),and the cells can be drawn into an injection pipette. The pipette isthen aspirated a few times to lyse the cells and remove cytoplasmiccomponents from the nucleus which is then injected into the oocyte. Thereconstituted oocytes are then activated, cultured, and transplantedinto maternal recipient mammals using standard methods such as thosedescribed herein to produce cloned mammals.

EXAMPLE 7 Evidence for More Complete Nuclear Reprogramming Using TwoNovel Cloning Procedures: Chromatin Transfer (CT) and StreptolysinO-Transfer (SLOT)

As illustrated above, incomplete nuclear remodeling and reprogrammingoccurs in traditional nuclear transplant pronuclear stage embryos. Thisfinding was demonstrated by the assembly of lamins A/C in the nuclearenvelope of pronuclear nuclear transplant embryos and excess NuMAimmunofluorescence labeling. More complete nuclear reprogramming wasachieved using the chromatin mass transfer method and the cellpermeabilization and reprogramming method (also referred to as SLOT)described herein.

In particular, the cloning methods of the present invention producedembryos with protein expression patterns that more closely resembled invitro fertilized embryos than cloned embryos produced using traditionalcloning methods. As described herein, chromatin transfer embryosexpressed much less lamin A/C protein than traditional nuclear transferembryos. Lamins A/C are somatic-specific components of the nuclearlamina that are naturally expressed in differentiated cells, but notexpressed in embryos. Because of the reported interaction of lamins withtranscription factors, chromatin proteins, and DNA, it is likely thatthe expression of lamins A/C in traditional nuclear transfer embryospromotes the expression of proteins specific for somatic cells that areundesirable for embryo development. Thus, the chromatin transfer embryosof the present invention may express fewer undesirable somatic-specificproteins than traditional nuclear transfer embryos. Additionally, thechromatin transfer embryos had expression patterns for NuMA, a maincomponent of the nuclear matrix that is implicated in transcriptionalregulation, that more closely resembled in vitro fertilized embryos thantraditional nuclear transplant embryos. This result also indicates thatchromatin transfer embryos are more efficiently reprogrammed thantraditional nuclear transplant embryos.

Assessment of In Vitro Nuclear Breakdown of Bovine Fibroblast NucleiIncubated in a Mitotic Extract and Characterization of the ResultingChromatin Masses Extracts prepared from mitotic bovine fibroblastsconsistently supported breakdown of ˜80% of input purified fibroblastnuclei (FIG. 33). An extract from metaphase II oocytes (i.e., an extractfrom oocytes naturally arrested in metaphase II prior to fertilization)also successfully supported nuclear breakdown (75% of nuclei within 30minutes).

Input interphase nuclei (FIG. 34A), chromatin masses obtained fromnuclei incubated in a MS15 mitotic extract (FIG. 34B), and chromatinmasses obtained from nuclei incubated in an oocyte extract (FIG. 34C)were examined for the expression of the following markers: lamin Breceptor (LBR), an integral protein of the inner nuclear membrane(membrane marker); lamin B, a ubiquitous component of the nuclearlamina; lamins A/C, a somatic-specific component of the nuclear laminapresent only in differentiated cells and absent in embryos; NuMA, a maincomponent of the nuclear matrix; AKAP95, a PKA-anchoring protein of thenucleus; and DNA. Both somatic cytosolic MS15 and oocyte MS15 extractsinduced solubilization of lamin B, lamins A/C, LBR, and NuMA in ˜100% ofchromatin units examined (FIGS. 34B and 34C). As expected, AKAP95remained associated with chromosomes, as observed previously in mitotichuman cells (Collas et al., J. Cell Biol. 147:1167-1180, 1999). Thisresult was also described herein for bovine nuclear transplant embryosat the premature chromatin condensation stage. Both the mitotic extractand the oocyte extract appeared to be as efficient as intact oocytes inpromoting nuclear envelope solubilization, regardless of the methodused, i.e., traditional nuclear transplant, nuclear injection (NI), orchromatin transfer (FIG. 35).

Comparison of Pronuclear Embryos Produced by Chromatin Transfer andPronuclei from Nuclear Transplant and Nuclear Injection Embryos Togenerate chromatin transfer embryos, in vitro-matured oocytes wereenucleated about 18-20 hours post maturation. Nuclei from interphasebovine fetal fibroblasts were incubated in a MS15 mitotic extract thatwas prepared from bovine fetal cells as described herein. Chromatinmasses were isolated from the extract when after nuclear envelopebreakdown had occurred and before chromatin condensation was completed.In particular, the chromatin masses were isolated when the chromatin wasapproximately 50-60% condensed, compared to the level of condensation ofchromosomes in interphase (designated 0% condensed) and the maximumlevel of condensation of chromosomes in mitosis (designated 100%condensed.) At this stage, individual chromosomes in the chromatin masscould not be distinguished and the edges of the chromatin mass had anirregular shape. Chromatin masses that had been isolated from themitotic extract were placed in a microdrop of TL HEPES with 2.5% sucrosealong with enucleated oocytes. The sucrose was added to the buffer tominimize damage to the oocytes from the subsequent injection procedure.Chromatin masses were injected into the oocytes using a beveledmicroinjection pipette using a Burleigh Piezo Drill (Fishers, N.Y.)(frequency 2 Hz for 75 microseconds at an amplitude of 70 V). Typicallymultiple pulses, such as 2, 3, 4, or 5 pulses, were performed so thatthe needle sufficiently penetrated the oocyte for injection. Afterinjection, oocytes were washed in serial dilutions of TL HEPES insucrose to minimize osmotic shock. At 28-30 hours post maturation (i.e.,28-30 hours after oocytes were placed in maturation medium aftercollection from ovaries, which is also at least two hours afterinjection of chromatin masses), reconstructed oocytes and controls forparthenogenetic development were activated with calcium ionophore (5 μM)for four minutes (Cal Biochem, San Diego, Calif.) and 10 μg/mlcycloheximide and 2.5 μg/ml cytochalasin D (Sigma) in ACM culture medium[100 mM NaCl, 3 mM KCl, 0.27 mM CaCl₂, 25 mM NaHCO₃, 1 mM sodiumlactate, 0.4 mM pyruvate, 1 mM L-glutamine, 3 mg/ml BSA (fatty acidfree), 1% BME amino acids, and 1% MEM nonessential amino acids (Sigma)],for five hours as described earlier (Liu et al., Mol. Reprod. Dev.49:298-307, 1998). After activation, eggs were washed five times andplaced in culture in four-well tissue culture plates containing mousefetal fibroblasts and 0.5 ml of embryo culture medium covered with 0.3ml of embryo tested mineral oil (Sigma). Between 25 and 50 embryos wereplaced in each well and incubated at 38.5° C. in a 5% CO₂ airatmosphere. If desired, calcium (e.g., ˜0.5, 1.0, 1.5, 2.0, 2.5, 3, 3.5,5 mM, or more CaCl₂) can be added to the culture medium for ˜0.5, 1.0,1.5, 2.0, 2.5, 3.0, or more hours to promote resealing of the oocyteafter injection. The resealed oocytes are likely to have increasedsurvival rates due to the intact layer surrounding the oocytes when theyare implanted into the recipient mammal using the standard methodsdescribed herein.

Nuclear injection embryos were formed as described above for chromatintransfer embryos, except that interphase bovine fetal fibroblasts nucleithat had not been incubated in an extract were injected into the oocytesinstead of chromatin masses. Nuclear transplant embryos were generatedusing the conventional methods described herein.

Nuclear transplant, nuclear injection, and chromatin transfer pronucleireassemble lamin B (FIG. 36A, red label) and AKAP95 (FIG. 36B, redlabel) as anticipated. Nuclear transplant and nuclear injectionpronuclei also reassemble lamins A/C, a somatic-specific component (FIG.36A, green label), consistent with the results reported above fornuclear transplant embryos. However, chromatin transfer pronuclei andcontrol parthenote pronuclei do not reassemble lamins A/C (FIG. 36A).Nuclear transplant pronuclei also contain NuMA (green label), unlikemost chromatin transfer or parthenote pronuclei (FIG. 36B, green label).A proportion of parthenote nuclei and chromatin transfer nuclei assemblea low level of NuMA, as reported above.

In vitro disassembly of nuclei followed by chromatin transfer results inpronuclei that are morphologically similar to control parthenotepronuclei. In contrast, nuclear transplant and nuclear injectionpronuclei harbor somatic-specific components (lamins A/C and extensiveNuMA labeling). This result is indicative of incomplete nuclearremodeling after traditional nuclear transplant or nuclear injectionprocedures. As described above, lamins A/C detected in nucleartransplant and nuclear injection pronuclei originate from laminstranscribed de novo at the pronuclear stage. Because nuclear lamins andpossibly NuMA are implicated in transcription regulation and disease inhumans, persistence of lamins A/C in conventional nuclear transplantpronuclei might be indicative of improper functional reprogramming. Weconclude that in vitro nuclear disassembly and chromatin transferproduces more normal pronuclei than traditional nuclear transplant ornuclear injection.

Cloning Efficiency using Reprogrammed Chromatin Masses or PermeabilizedCells as Donor Source As described herein, a novel cloning proceduredenoted “SLOT” was developed that involves Streptolysin O (SLO)-inducedpermeabilization of primary fetal bovine fibroblasts, exposure ofpermeabilized cells to a reprogramming media (e.g., a mitotic extract)for 30 minutes, optionally resealing of the fibroblasts with 2 mMcalcium in culture, and transfer of the chromatin into oocytes usingstandard cell fusion methods.

For this cloning method, a vial of Streptolysin O (Sigma S-5265; 25,000units stored in store powder form at 4° C.) was dissolved in 400 μl H₂Oand mixed well. All contents were transferred to a 15-ml conical tube,and then 3.6 ml H₂O was added and mixed by vortexing. Aliquots of 10 μlwere frozen at −20° C. at a stock concentration of 0.062 U/μl. Cells(˜100,000) were suspended in 100 μl HBSS (Gibco BRL, cat. No. 14170-120)at room temperature. These cells were confluent, and thus ˜80-85% of thecells were in G1 phase, and the majority of the other cells were in Sphase. Streptolysin O stock solution (5 μl) (i.e., 500 ng/ml or 0.3 U/μlfinal concentration) was added, and the mixture was incubated at 38° C.for 25 minutes in a water bath. The tube was gently tapped 2-3 timesduring incubation to ensure that the cells remained in suspension. Roomtemperature PBS (200 μl) was added and mixed well by gentle pipetting.The cells were centrifuged cells at 5,000 rpm for five minutes at roomtemperature in a table top centrifuge. All the supernatant wasdiscarded. At this stage, the pellet is small and may not be clearlyvisible. Mitotic extract containing the ATP-generating system (40 μl,“MS15”) was added and mixed well. The extract was prepared during thecentrifugation of the cells by thawing one vial of 40 μl extract andadding 1.2 μl of ATP-generating system, mixing well, and incubating atroom temperature. This mitotic extract was the same extract used for thegeneration of chromatin masses in the section above. The mixture wasincubated at 38° C. in water bath for 30 minutes, and the tube wasoccasionally gently tapped. Room temperature resealing medium (RM, 500μL) (complete α-MEM [Bio-Whittaker] medium supplemented with CaCl₂ to 2mM from a 1 M stock) was added. The tube was left open and incubated ina CO₂ incubator for two hours with occasional tapping of the tube toensure that the cells remained in suspension. The cells were centrifugedat 5,000 rpm for five minutes at room temperature in a table topcentrifuge. The cell pellet was resuspended in 100 μl of roomtemperature TL HEPES (Bio-Whittaker, cat. No. 04-616F), and another 900μl TL HEPES was added. The nuclear transfer was performed using standardprocedures. Oocytes were activated and transferred to recipient mammalsas described in the previous section for chromatin transfer.

The development of embryos formed using this SLOT method and thechromatin transfer method of the present invention is summarized inTable 7. Development to the blastocyst stage was slightly lower for SLOTembryos compared to conventional nuclear transfer embryos. Thedifferences between SLOT and nuclear transfer development at theblastocyst stage could be due to the effect of using a greaterpercentage of cells in the G1 phase of the cell cycle for nucleartransfer than for SLOT. The survival rate was lower for chromatintransfer embryos, which is expected for an invasive procedure.

Pregnancy rates were comparable for nuclear transfer and SLOT embryos at40 days of gestation (Table 7). Survival from 40 days of pregnancy to 60days tended to be higher for SLOT embryos than for nuclear transferembryos produced using conventional methods. TABLE 7 Development ofchromatin transfer (CT), nuclear transplant, and SLOT- produced bovineembryo clones No. No. No. No. Survived No. Survived PN stage No. CleavedBlastocysts No. 40 day 40-60 days/total transferred (%) (%) (%) (%)Preg. (%) (%) CT 1503 736  (49) 355 (23.5) 81 (5.3) 3 0 ND SLOT 18841802 (97) ND 575 (30.5) 156 (8.3)  24/65 (37) 7/10 (70) nuclear 18211682 (92) ND 764 (41.9) 235 (12.9) 39/103 (36)  8/16 (50) transplant

As noted above, the survival rate for chromatin transfer embryos may beincreased by incubating the reconstituted oocytes in calcium for a fewhours to allow the oocytes to reseal prior to be inserted into recipientmammals. Survival rates for SLOT embryos may also be increased byreducing the amount of time between when the cells are taken out ofculture and when they are fused with oocytes. For example, the length oftime for the incubation in Streptolysin O, the incubation in thereprogramming medium, and/or the incubation in the resealing medium maybe decreased. In particular, the incubation in the resealing medium maybe decreased to approximately one hour or less. This shortened resealingtreatment may be performed in the presence of 2 mM calcium as describedabove or in the presence of a higher concentration of calcium (e.g.,˜2.5, 3.0, 3.5, 4.0, 4.5, 5.0, or 6.0 mM calcium) to increase the rateof resealing. By reducing the amount of time the cells are treated priorto being fused with oocytes, the cells are less likely to enter S phaseand begin DNA replication which reduces the survival rate of thereconstituted oocyte.

EXAMPLE 8 Evidence for More Complete Nuclear Reprogramming UsingChromatin Transfer (CT)

As discussed above, a strategy was developed to enhance remodeling ofdonor nuclei, promote repression of somatic genes, and produce embryoswith a pronuclear architecture similar to that of fertilized zygotes. Ina particular example of this general chromatin transfer method, isolatedintact bovine fibroblast nuclei were incubated in a cytoplasmic extractof mitotic bovine fibroblasts in the presence of an ATP-generatingsystem, which was required to drive nuclear disassembly.

To generate a mitotic reprogramming extract for conversion of donornuclei into chromatin masses, fibroblasts were synchronized in mitosiswith 0.5 μg/ml nocodazole for 18 hours, harvested by mitotic shake-off,and washed twice in ice-cold PBS and once in ice-cold cell lysis buffer(20 mM Hepes, pH 8.2, 5 mM MgCl₂, 10 mM EDTA, 1 mM DTT, and proteaseinhibitors) (Collas et al., J. Cell Biol. 147:1167-1179, 1999). Packedcells were resuspended one volume of cell lysis buffer, allowed to swellon ice for one hour and Dounce-homogenized on ice using a tight fittingpestle until all cells were lysed. The lysate was centrifuged at15,000×g for 15 minutes at 4° C., and the supernatant (mitotic extract)was collected, aliquoted, and snap-frozen in liquid nitrogen and storedat −80° C.

To generate donor chromatin, unsynchronized confluent fibroblasts wereharvested, washed, and resuspended in ˜20 volumes of ice-cold hypotonicnuclear isolation buffer (10 mM Hepes, pH 7.5, 2 mm MgCl₂, 25 mM KCl, 1mm DTT, and a cocktail of protease inhibitors) (Collas et al., J. CellBiol. 147:1167-1179, 1999). After one hour on ice, cells wereDounce-homogenized with a tight-fitting pestle. Sucrose was added from a2 M stock to a concentration of 250 mM and nuclei sedimented at 400×gfor 10 minutes at 4° C. Nuclei were washed in nuclear isolation buffer(same buffer as above but with 250 mM sucrose) and were either usedfresh or frozen in nuclear isolation buffer/70% glycerol (Collas et al.,J. Cell Biol. 147:1167-1179, 1999).

For reprogramming, isolated fibroblast nuclei were incubated in 40 μl ofmitotic extract containing an ATP-generating system (1 mM ATP, 10 mMcreatine phosphate, and 25 μg/ml creatine kinase) at 4,000 nuclei/μl for30 minutes in a 38° C. H₂O bath. Nuclear envelope breakdown andchromatin condensation were monitored by phase contrast microscopy. Atthe end of incubation, the reaction mix was diluted with 500 μl TL Hepescontaining 2.5% sucrose, and chromatin was recovered by sedimentation at2,000×g for five minutes. Chromatin masses were resuspended in TLHepes/sucrose and transferred to TL Hepes/sucrose under mineral oiltogether with enucleated oocytes. Individual chromatin masses wereinjected into in vitro-matured oocytes enucleated at 20 hpm with abeveled microinjection pipette using a Burleigh Piezo Drill (Fishers,N.Y.) (2 Hz, 2 μs, 70 V). After injection, oocytes were washed in serialdilutions of sucrose in TL Hepes to minimize osmotic shock and cultured.At 28 hpm, reconstructed oocytes and parthenogenetic controls wereactivated as described for NT and cultured ((Kasinathan et al., Nat.Biotechnol. 19:1176-1178, 2001 and Kasinathan et al., Biol. Reprod.64:1487-1493, 2001). For nuclear injections (NI), purified fibroblastnuclei were exposed to cell lysis buffer instead of mitotic extract andinjected into enucleated oocytes as for CT.

As a result of reprogramming, lamin A/C, lamin B, and NuMA were readilydisassembled, while AKAP95 remained associated with condensedchromosomes. TUNEL analysis showed that no apoptosis occurred in theextract. Similar nuclear breakdown studies with human HeLa nuclei andmitotic extracts indicated that condensed chromatin masses were capableof supporting nuclear reconstitution (Steen et al., J. Cell Biol.150:1251-1562, 2000) and transcription in interphase cytoplasm. Thus, invitro nuclear disassembly produces condensed chromatin capable ofreforming functional nuclei. Condensed fibroblast chromatin wasrecovered by sedimentation, and individual chromatin masses wereinjected into enucleated recipient oocytes. After recovery in culture,oocytes were activated as described herein for NT oocytes. To controlfor artifacts generated by handling of nuclei, intact nuclei exposed toextract buffer alone were also injected.

NI produced pronuclei which, like NT pronuclei, contained lamins A/C andB, NuMA, and AKAP95. CT resulted in PN formation in over 80% of embryosthat survived injection and activation (˜50% of oocytes injected,n>2,000). However, CT pronuclei displayed no detectable lamin A/C and a3-fold reduction of anti-NuMA immunolabeling compared to NT and NIpronuclei. This pattern was similar to that of parthenogeneticpronuclei. Extractability of AKAP95 and DNA with Triton X-100/DNAseI/NaCl as described above was enhanced 8-fold in CT pronuclei comparedto NT pronuclei, reflecting a beneficial effect of CT on pronuclearAKAP95 anchoring and DNAse I accessibility. As chromatin-bound AKAP95co-fractionates with primarily transcriptionally repressed(hypoacetylated) chromatin, this result suggests that CT enhancesformation of euchromatin upon PN assembly.

The dynamics of the TATA binding protein, TBP, was examined duringfibroblast donor nucleus remodeling by NT and CT procedures carried outin parallel. TBP facilitates assembly of the general transcriptionmachinery for virtually all genes (Sharp et al., Cell 68:819-821, 1992).TBP co-localized with AKAP95 and DNA in donor fibroblast nuclei. PCCchromosomes obtained after NT contained 5-fold more TBP than chromosomescondensed in mitotic extract, as shown by TBP/AKAP95 and TBP/DNAfluorescence intensity ratios. TPB labeling intensity of chromosomescondensed in vitro resembled that of mitotic fibroblasts. At the PNstage, TBP was barely detectable in CT embryos but displayed strongimmunoreactivity in NT and NI embryos. Pronuclear TBP in NT embryos wasof somatic origin because inhibition of transcription or translationmaintained strong TBP labeling. Pronuclear TBP concentration in themouse has been shown to increase during progression through interphase(Worrad et al., Development 120:2347-2357, 1994). However, thesimilarity in kinetics of PN formation from PCC- or in vitro-condensedchromatin between reconstructed NT and CT embryos indicated that theenhanced TBP concentration in NT pronuclei was not due to more aadvanced cell cycle stage. Thus, in vitro disassembly of donor nucleipromotes dissociation of TBP from the chromatin, such that resulting CTpronuclei contain ˜10-fold less TBP than NT pronuclei at the same stageof reconstitution.

Thus, five structural and functional markers of incomplete reprogrammingby NT include pronuclear assembly of lamins A/C, enhanced pronuclearNuMA and TBP concentrations, and increased sensitivity of AKAP95 and DNAto extraction with detergent, DNAse, and salt. In contrast, B-typelamins, essential for proper nuclear reformation and cell survival(Steen et al., J. Cell Biol. 153:621-626, 2001) appear to assemblenormally in NT pronuclei, although we were not able to determine whetherthe pool of assembled B-type lamins was of somatic (and thereforere-targeted) or of maternal origin. The LMNA gene remains active in NTpronuclei, resulting in the assembly of differentiated cell-specificA-type lamins. Through interactions with chromatin and the transcriptionmachinery, nuclear lamins have been suggested to participate intranscription regulation (Cohen et al., Trends Biochem. Sci. 26:41-47,2001); thus, assembly of the correct set(s) of nuclear lamins is mostlikely critical for proper pronuclear function in NT embryos.

In the present methods, somatic nuclear components are dispersed in theextract and typically do not come in contact with the oocyte cytoplasm.This prohibits re-targeting of somatic-specific molecules to thedeveloping nucleus, such as B-type lamins whose composition may differfrom that of maternal lamins (Cohen et al., Trends Biochem. Sci.26:41-47, 2001). In vitro chromatin condensation may also promoterelease of DNA-bound factors such as chromatin remodeling enzymes (Sifet al., Genes Devel. 12:2842-2851, 1998) and transcription factors,thereby “stripping” the donor genome of potentially inhibitory somaticcomponents. In particular, TBP removal may result in inactivation ofsomatic-specific genes in reconstituted CT pronuclei and duplicate thelow transcriptional activity of the male pronucleus after fertilization(Poccia et al., Trends Biochem. Sci. 17:223-227, 1992).

An implication of removing factors from the donor nucleus is thatloading of maternal components onto chromatin and remodeling into aphysiological pronucleus may be facilitated. CT increases thesensitivity of AKAP95 and DNA to nucleases and salt. DNAse I-resistantDNA is mostly transcriptionally silent; thus, incomplete remodeling ofAKAP95 anchoring by NT may impair expression of developmentallyimportant genes, such as genes involved in placental development,maintenance of late pregnancy and post-natal survival of cloned animals.

The following characteristics of CT oocytes indicate that nucleartransfer of a chromatin mass incubated in a mitotic extractsignificantly improves the functional characteristics of the resultingreconstituted oocyte. A nuclear matrix protein that is expressed at highlevels by somatic donor cells, NuMA, was expressed 3-fold less in CTpronuclei (i.e., pronuclei formed after introduction of a chromatin massinto an oocyte) than in NT pronuclei. This result indicates that thelevel of NuMA in CT oocytes is more similar to the level of NuMA innaturally-occurring oocytes than in NT oocytes. CT pronuclei alsoexpressed 10-fold less of the general transcription factor TBP than NTpronuclei. This removal of TBP from the donor chromatin mass byincubation in the mitotic extract may result in inactivation ofundesired, somatic-specific genes in the resulting CT oocyte. Lamin A/C,a nuclear envelope protein that is specific for differentiated cells andis not detected in in vitro fertilized or parthenogenically activatedoocytes, was also not detected in CT pronuclei but was detected in NTpronuclei. Because nuclear lamins may regulate transcription, the lackof detectable lamin A/C in CT pronuclei may result in more appropriateregulation of transcription in CT oocytes than in NT oocytes. Thesensitivity of AKAP95, a structural protein of the nuclearmatrix-chromatin interface which is enriched in transcriptionallyrepressed (hypoacetylated) chromatin, and DNA to extraction withdetergent, DNase, and salt was measured in the pronuclei to characterizethe anchoring of AKAP95 to DNA and to characterize DNA accessibility inthe pronuclei. Extractability was enhanced 8-fold in CT pronucleicompared to NT pronuclei, reflecting a beneficial effect of incubationof a donor chromatin mass in a mitotic extract on AKAP95 anchoring andmorphological remodeling of the donor DNA. Because AKAP95 and DNaseI-resistant DNA is associated with transcriptionally repressed or silentDNA, the increased sensitivity of AKAP95 and DNA to nucleases, salt, anddetergent suggests that CT oocytes may have increased expression ofdevelopmentally important genes.

Similar results are expected with donor nuclei from cells with one ormore mutations in an endogenous immunoglobulin or prion gene.

EXAMPLE 9 Evidence for More Complete Nuclear Reprogramming UsingStreptolysin O-Transfer (SLOT)

The reprogramming of a chromatin mass during incubation of apermeabilized cell in a mitotic extract using SLOT was alsodemonstrated. In this study, the expression of a nuclear matrix proteinthat is expressed at high levels by somatic donor cells, NuMA, and theexpression of a nuclear envelope protein that is specific fordifferentiated cells, lamin A/C, was compared for oocytes produced usingSLOT and oocytes produced using traditional nuclear transfer of a cellcontaining a nucleus that has not been incubated in an extract (“NT”).Fourteen hours after activation, the SLOT pronuclei (i.e., pronucleiformed after introduction of a donor cell containing a chromatin massinto an oocyte) expressed significantly less NuMA and lamin A/C than NTpronuclei (n=15-20 embryos/marker in three replicates). These resultsdemonstrate that reconstituted SLOT oocytes are more efficientlyreprogrammed than NT oocytes and more closely resemblenaturally-occurring, fertilized oocytes. Because nuclear lamins mayregulate transcription, the significantly lower level of lamin A/C inSLOT pronuclei may result in more appropriate regulation oftranscription in SLOT oocytes than in NT oocytes.

In addition to reducing the expression of undesired factors in theresulting oocyte, SLOT increases the viability of the resulting fetusescompared to traditional nuclear transfer methods (Table 8). Thus, themany structural and functional differences of SLOT donor cellssubstantially improve the ability of the reconstituted oocytes to formnon-human embryos and non-human mammals. TABLE 8 Development of bovineembryos produced using permeabilized donor cells with a chromatin mass(“SLOT”) compared to bovine embryos produced using donor cells with anucleus (“NT”) No. No. of No. 40 day No. 90 day transferred RecipientsPreg. (%) Preg. (%) SLOT 1955 59 29/59 (49) 14/59 (24) NT 1885 95 44/95(46) 16/95 (17)

Similar results are expected with donor cells with one or more mutationsin an endogenous immunoglobulin or prion gene.

EXAMPLE 10 Exemplary Evidence for More Complete Nuclear ReprogrammingUsing Streptolysin O-Transfer (SLOT)

As described above, a novel in vitro nuclear remodeling system has beendeveloped. The system involves permeabilization of the donor cell,induction of chromatin condensation in a mitotic cell extract, andwashing of the permeabilized cell to remove nuclear factors solubilizedduring condensation of the chromatin. Pronuclei of bovine chromatintransplant embryos exhibit an expression pattern of several markers thatclosely resembles that of normal embryos as opposed to nucleartransplant embryos, which resemble somatic cells. Eight healthy calveswere produced using this chromatin transfer system. Chromatin transfershows trends of increased survival to term, lower incidence of largecalves, and significantly enhanced survival after birth. The resultsdemonstrate the successful manipulation of a somatic donor nucleus priorto transplantation. As described further below, the disassembly of asomatic nucleus in a mitotic extract followed by transfer of thecondensed chromatin into an oocyte enhances nuclear remodeling and showsevidence of improved development and viability of clones. This procedurecan be used to further characterize the mechanism of nuclearreprogramming, if desired.

A chromatin transfer strategy To alleviate defects identified inpronuclear NT embryos, fibroblast nuclei were manipulated in vitro priorto transfer into recipient oocytes. This SLOT system is outlined in FIG.45. For generation of a mitotic extract, fibroblasts were synchronizedin mitosis with 1 μg/ml nocodazole for 18 hours, harvested by mitoticshake-off, and washed twice in phosphate buffered saline and once incell lysis buffer (20 mM Hepes, pH 8.2, 5 mM MgCl₂, 10 mM EDTA, 1 mM DTTand protease inhibitors). Sedimented cells were resuspended in onevolume of ice-cold cell lysis buffer, allowed to swell on ice for onehour, and Dounce-homogenized using a tight-fitting glass pestle. Thelysate was centrifuged at 15,000×g for 15 minutes at 4° C. and thesupernatant (mitotic extract) was aliquoted, frozen in liquid nitrogen,and stored at −80° C. Fresh or frozen extracts were used withoutnoticeable differences on efficiency of nuclear breakdown.

Bovine fetal fibroblasts from confluent cultures were washed inCa²⁺/Mg²⁺-free Hank's Balanced Salt Solution (HBSS) and permeabilized byincubation of 100,000 cells in suspension with 31.2 U Streptolysin O(SLO; Sigma) in 100 μl HBSS for 30 minutes in an approximately 38.5° C.H₂O bath. Permeabilization was assessed by uptake of the membraneimpermeant DNA stain, propidium iodide (0.1 μg/ml). Permeabilizedfibroblasts were sedimented, washed, and incubated in 40 μl mitoticextract containing an ATP-generating system (1 mM ATP, 10 mM creatinephosphate, and 25 μg/ml creatine kinase) for 30-45 minutes atapproximately 38.5° C. to promote nuclear disassembly and removal ofnuclear components. Aliquots were labeled with 0.1 μg/ml Hoechst 33342to monitor chromatin condensation. After the incubation, fibroblastswere recovered from the extract by sedimentation and washed. Thereaction mixture was diluted with 500 μl Alpha MEM/10% fetal bovineserum (Gibco-BRL) containing 2 mM CaCl₂ for membrane resealing, and thecells were cultured for two hours at 38.5° C. (H

kelien et al., Nat. Biotechnol. 20:460-466, 2002). Resealing wasmonitored by propidium iodide uptake. Resealed cells were fused with invitro-matured oocytes that had been enucleated at 20 hpm; oocytes wereactivated at 28 hpm, and embryos cultured as described herein for NT.

SLOT embryos were cultured to the blastocyst stage in vitro, and twoembryos were transferred per recipient female. Pregnancies weremonitored by ultrasonography, and C-sections were performed. Calves werescored by veterinarians within 24 hours of birth.

Breakdown of fibroblast nuclei in mitotic extract The mitotic extractconsisted of a 15,000×g supernatant from a lysate of mitotic bovinefibroblasts and contained an ATP-regenerating system. The extract didnot induce apoptosis, as judged by the absence of proteolysis ofpoly(ADP)ribosyl polymerase (PARP) and DNA fragmentation characteristicof apoptotic fibroblasts. Thus, the extract was suitable to promoteremodeling of somatic nuclei.

The extract elicited ATP-dependent condensation of chromosomes,disassembly of A/C and B-type lamins from chromatin as judged bycytoplasmic labeling of these lamins, and removal of TBP from chromatin.These events were confirmed by immunoblotting analysis of condensedchromatin purified from the fibroblasts after recovery from the mitoticextract. In this experiment, the A-kinase anchoring protein AKAP95 wasused as a marker of a nuclear component that remains associated with thecondensed chromosomes, as normally occurs at mitosis (Collas et al., J.Cell Biol. 147:1167-1180, 1999). Histone H4 was used as a proteinloading control in the gel. Disassembly of nuclear lamins and TBP fromchromatin in mitotic extract was dependent on an ATP-regenerating systemand was reminiscent of that occurring in mitotic cells. Furthermore,immunoblotting analysis of whole permeabilized fibroblasts (as opposedto isolated chromatin) after exposure to mitotic extract showed that afraction of solubilized lamin A/C and all detectable TBP were eliminatedfrom the cells and/or proteolysed. Lastly, a control extract frominterphase fibroblasts or cell lysis buffer alone both containing anATP-regenerating system failed to promote nuclear disassembly,indicating that nuclear breakdown was ATP-dependent and specific for themitotic extract. Permeabilized fibroblasts exposed to mitotic extractand resealed with CaCl₂ could be cultured over several passages,indicating that membrane permeabilization, incubation of thepermeabilized cells in the extract, and membrane resealing were viableprocedures.

Characterization of nuclei in embryos produced by chromatin transferFusion of resealed fibroblasts to recipient oocytes occurred asefficiently (over 70%) as with non-permeabilized cells. The donorchromatin was in a condensed form at the time of introduction intooocytes. In contrast, chromatin of fibroblasts used for NT was stilldecondensed within 30 minutes of fusion. Thus, resealing of mitoticextract-treated fibroblasts with CaCl₂ prior to transfer into oocytesdid not promote nuclear reformation in the donor cells. This observationwas supported by the absence of a nuclear envelope around the condensedchromatin in SLOT embryos immediately after fusion, as judged byimmunofluorescence analysis of several lamina and inner nuclear membraneproteins.

Immunolabeling of nuclear lamins and TBP in nuclei of SLOT and NTembryos and immunolabeling intensity of these markers relative to DNAfluorescence intensity was performed. Perinuclear lamin B labelingintensity was similar in SLOT and NT pronuclei. Remarkably however, incontrast to NT pronuclei, lamin A/C was undetected in pronuclei and upto at least the 8-16-cell stage in SLOT embryos. SLOT pronuclei alsodisplayed a 4-fold reduction in TBP labeling compared to NT pronuclei.Pronuclear TBP concentration in the mouse has been shown to increaseduring progression through interphase (Worrad et al., Development120:2347-2357, 1994). However, as kinetics of pronuclear formation fromPCC- or in vitro-condensed chromatin were similar in NT and SLOTembryos, it is unlikely, albeit not formally excluded, that enhanced TBPconcentration in NT pronuclei was due to a more advanced cell cyclestage. Resistance of TBP to extraction with 1% Triton X-100, 1 mg/mlDNAse I, and 0.3 M NaCl was decreased by over 2-fold, indicating aweaker association of TBP with intranuclear ligands. Likewise,resistance of DNA to DNAse I was reduced nearly 4-fold in SLOTpronuclei, suggesting that SLOT favors the establishment of a looserchromatin configuration in pronuclei. Thus, disassembly of fibroblastnuclei in mitotic extract followed by transfer of the condensedchromatin into oocytes enhanced morphological remodeling of the donornuclei and alleviated defects detected in pronuclei of NT embryos.

Chromatin transfer produces healthy clones SLOT resulted in developmentto term of cloned embryos. Pregnancy rates following transfer ofblastocysts into recipient females were significantly higher at 40 daysfor SLOT embryos than for NT embryos (P=0.02; Fisher's Test), and thetrend was maintained up to development to term (12/59 and 23/211 calvesborn, respectively; P=0.05). Additionally, SLOT enhanced survival rateof clones beyond 24 hours post-partum (10/59 and 17/211, respectively;P=0.04).

Health of NT and SLOT clones born was evaluated by scoring animals andplacentas on a scale of 1 (normal) to 5 (grossly abnormal). Placentalscores included parameters such as placental edema, cotyledon number,size and morphology, color of amniotic fluids, morphology of uterus, andumbilicus. Animal scores included functional evaluation and generalappearance of respiratory, cardiovascular, digestive, urinary, muscular,skeletal and nervous systems. Box plot analyses show that scores ofanimals and placentas derived from NT were more dispersed that thoseproduced by SLOT. Mean birth weight of SLOT and NT clones was notsignificantly different; however, the proportion of SLOT calves over 45kg at birth was lower (P=0.02; Fisher) than that of NT animals.Altogether, these results indicate that chromatin transfer produces liveoffspring and shows evidence of improved development and viability.

In comparison with NT and despite the limited number of SLOT offspringproduced (n=8), SLOT significantly enhances pregnancy rates at 40 days(P=0.02) and survival of calves born beyond 24 hours (P=0.04). A trendtowards an improvement in the health of animals produced by SLOT is alsoreflected in box plot analyses. Furthermore, the lower incidence oflarge (over 45 kg) calves produced by SLOT has practical and economicalimplications on animal management.

Several nuclear defects have been identified in NT embryos, includingassembly of lamin A/C, enhanced pronuclear TBP content and increasedresistance of DNA to DNAse I. These defects may result from incompleteremodeling of the fibroblast nuclei and/or from misregulation ofexpression of differentiated cell-specific (e.g., lamin A) genes.Remodeling of nuclei in vitro and transplantation of condensed chromatininto oocytes alleviates these defects.

Remodeling nuclei through SLOT increases DNA sensitivity to DNAse I andmay promote the formation of transcriptionally active (or potentiallyactive) chromatin. This effect may in turn facilitate expression ofdevelopmentally important genes, such as genes involved in placentaldevelopment, maintenance of late pregnancy, and post-natal survival.SLOT also induces repression of lamin A gene expression in clonedembryos. In vitro and in vivo manipulations of nuclear laminacomposition have shown that failure to assemble a correct set of laminsinvariably leads to apoptosis (Steen et al., J. Cell Biol. 153:621-626,2001). Moreover, as lamins interact with DNA, chromatin and thetranscription machinery, proper lamina reconstitution is likely to beessential for normal nuclear function (Gruenbaum et al., J. Struct.Biol. 129:313-323, 2000 and Cohen et al., Trends Biochem. Sci. 26:41-47,2001) in cloned embryos.

Chromatin condensation at mitosis or in vitro is associated with therelease of DNA-bound components such as chromatin remodeling enzymes,transcription factors (e.g., TBP), or other potentially inhibitorysomatic components. Removal of TBP from donor somatic chromatin mayfacilitate repression or down-regulation of somatic-specific genes inSLOT embryos, which may impair development. An implication of removingfactors from the donor nucleus is that loading of maternal componentsonto chromatin and subsequent remodeling into a physiological pronucleusmay be facilitated.

In conclusion, it is possible to directly remodel a somatic nucleus in acell extract and produce live offspring. In vitro manipulation of nucleifor cloning or transdifferentiation purposes (Landsverk et al., EMBORep. 3:384-389, 2002 and H

kelien et al., Nat. Biotechnol. 20:460-466, 2002) is a useful tool foroptional further investigation of the mechanisms of nuclearreprogramming. Chromatin transfer shows evidence of improved developmentto term and viability of clones. Additional manipulation of the systemmight lead to further improvements in the efficiency of mammaliancloning.

EXAMPLE 11 Additional Evidence for More Complete Nuclear ReprogrammingUsing Streptolysin O-Transfer (SLOT)

The SLOT method described above produced improved results when the donorpermeabilized cells were are not resealed prior to genetic transfer.Additionally, use of donor cells in G₁ phase instead of confluent cellsmay result in increased viability of the reconstituted oocyte andresulting embryo. These experiments are described further below.

Preparation of buffers for experiments below A 1 molar DTT solution wasprepared by placing 1 ml H₂O in an eppendorf tube, adding 154 mgrefrigerated DTT, and mixing well by vortexing. The solution wasaliquoted into 10 μl volumes and frozen at −20° C. Aliquots can bethawed and re-frozen several times. A 100 ml volume of cell lysis bufferfor preparation of cell extracts for nuclear assembly/disassembly assayscontained NaCl (50 mM, 1 ml of 5 M stock), MgCl₂, (5 mM, 0.5 ml of 1 Mstock), Hepes, pH 8.2 (20 mM, 2 ml of 1 M stock, pH 8.2), and H₂O (96.5ml). The buffer was aliquoted and stored at 4° C. There was a drop of ˜1pH unit upon lysate preparation. For preparation of mitotic extracts, 10mM EGTA was added (1 ml of a 1 M stock) and 95.5 ml H₂O was addedinstead of 96.5 ml. Prior to use, the following were added: DTT (1 μl/mlsolution, 1 M stock at −20° C., 1 mM final concentration), PMSF (10μl/ml solution 100 mM stock, 1 mM final concentration), CAL mix (10 μlCAL cocktail at −20° C. per ml solution, i.e., a final concentration of10 μg/ml each of chymostatin, aprotinin, and leupeptin), Pepstatin A (10μl stock at −20° C. per ml solution, 10 μg/ml final concentration), andCytochalasin D (1 μl/ml from 1 mg/ml stock at −20° C., 1 μg/ml finalconcentration). For the nocodazole 1000× stock solution, 1 mg/ml ofnocodazole in DMSO was prepared and stored in 160 μl aliquots.

To prepare Streptolysin O stock (SLO), a vial of SLO (Sigma S-5265;25,000 units stored as a powder at 4° C.) was dissolved in 400 μl H₂Oand mixed well. The entire content was transferred to a 1.5-ml conicaltube, divided into 10 μl aliquots, and frozen at −20° C. The stockconcentration was “10×.” To prepare the protease solution, 3 ml TL Hepesand 9 mg Protease (Sigma P-8811) were added to a 15 ml conical tube andmixed by vortexing. The solution was filtered through a 0.22 um syringefilter directly into TL Hepes. Because the cells doubled the volume, thefinal concentration was 1.5 mg/ml. For HECM Hepes, NaCl (114 mM, 6.662g), KCl (3.2 mM, 0.239 g), CaCl₂ 2H₂O (2.0 mM, 0.294 g), MgCl₂ 6H₂O (0.5mM, 0.102 g), Pen/Strep (10 ml, Sigma P3539 Pen/Strep, 100 U/ml and 100ug/ml final concentration), Phenol Red (5 ug/ml, 1 ml), and H₂O (in asufficient amount to increase the total volume to 990 ml) were combined.Then, 100×A.A. (10 ml), Na lactate (10 mM, 1.44 ml), Na pyruvate (0.1mM, 0.011 g), NaHCO₃ (2 mM, 0.168 g), and HEPES (10 mM, 2.38 g) wereadded. The final solution had an osmolarity of 260-270 mOsM. Then, 3 gbovine serum albumin (Fraction V) was added, and the pH was adjusted to7.4. The solution was filtered through a 0.22 uM filter and stored at 4°C.

For preparation of the ATP stock solution (Sigma A3377: 100 mM Stock,100×), H₂O (1 ml) and ATP (0.055 g) were combined, and 10 μl aliquotswere frozen at −20° C. For preparation of creatine phosphate (SigmaP7936: 1 M stock, 100×), H₂O (1 ml) and creatine phosphate (0.255 g)were combined, and 10 μl aliquots were frozen at −20° C. To preparecreatine kinase (Sigma C3755: 2.5 mg/ml stock, 100×), H₂O (1 ml) andcreatine kinase (0.0025 g) were combined, and 10 μl aliquots were frozenat −20° C. For preparation of the ATP-generating system, equalproportions of 100 mM ATP stock, 1 M creatine phosphate, and 2.5 mg/mlcreatine kinase stock solutions (100×) were made using H₂O, mixed, andstored as a frozen solution. The ATP generating system was kept on iceuntil use. The ATP generating system (1.2 μl) was added to the extract(40 μl), and the solution was mixed by vortexing. The finalconcentration of the ATP generating system in the extract was 1 mM ATP,10 mM creatine phosphate, and 25 μg/ml creatine kinase.

Preparation of mitotic extract One vial of 1.5 to 2 million cells wasthawed. Cells were split into two T75 flasks and grown for two days oruntil they were confluent. The cells were passaged in 6-8 T75 flasks andgrown to confluency. The cells were trypsinized and counted using ahemocytometer, and 3 million cells were added to each of as many T150flasks as could be used with the number of cells available. The cellline (e.g., fibroblasts such as primary fibroblasts, epithelial cells,or immortalized and disease-free cells such as MDBK cells) wassynchronized at 70-80% confluency in mitosis with 0.5-1 μg/ml nocodazolefor 17-20 hours using standard procedures (e.g., Collas et al., J. CellBiol. 147:1167-1180, 1999 and references therein). The synchronizedcells were harvested by mitotic shake-off. Each flask containing cellswas shaken vigorously by repeatedly tapping it with one hand. Themitotic cells detached and floated in the culture medium. The harvestedcells were centrifuged at 500×g for 10 minutes in a 50 ml conical tubeat 4° C. The supernatant was discarded, and the cell pellets wereresuspended in a total of 50 ml of cold phosphate buffered saline/Ca/MgFree (PBS). If desired, several cell pellets can be pooled into a single50 ml tube. The cells were centrifuged at 500×g for 10 minutes at 4° C.,and the above washing step was repeated. The volume of the cell pelletwas determined, and the cell pellet was resuspended in approximately 20volumes of ice-cold cell lysis buffer containing protease inhibitors(i.e., DTT and PMSF).

Then, the cells were sedimented at 500×g for five minutes at 4° C. Thesupernatant was discarded, and the cell pellet volume was determined.The cell pellet was resuspended in no more than one volume of cell lysisbuffer containing all of the protease inhibitors. The cells wereincubated on ice for one hour to allow swelling of the cells. Using atip sonicator, the cell suspension was sonicated until all cells werebroken open. Cell lysis was monitored under a phase contrast microscope.Desirably, 90% of the cells were lysed before proceeding to the nextstep. Sonication can be prolonged as long as necessary; the sonicationtime varies with the cell type used to prepare the extract.Alternatively, cells are lysed by Dounce homogenization using a glassmortar and pestle (homogenizer), desirably until at least 90% of thecells are lysed. The cell lysate was placed in a 1.5-ml centrifuge tubeand centrifuged at ˜15,000×g for 15 minutes at 4° C. using a table toprefrigerated centrifuge. The centrifuge was placed in a cold room orrefrigerator and allowed to equilibrate. The tubes were removed from thecentrifuge and immediately placed on ice. The supernatant was carefullycollected using a 200 μl pipette tip. This supernatant is the mitoticcytoplasmic extract. The extract was placed in another tube on ice.Extracts collected from several tubes were pooled.

Two alternatives exist at this stage. The cell extract was aliquotedinto tubes on ice, 41 μl of extract per tube. The extract wasimmediately snap-frozen in liquid nitrogen and stored in a −80° C.freezer until use. Such extracts prepared from a 15,000×g centrifugationare called MS15 (or mitotic cytoplasmic extract). Alternatively, theMS15 extract is placed in an ultracentrifuge tube on ice (e.g., fittedfor an SW55 Ti rotor; Beckman). The tube is overlayed with mineral oilto the top if necessary to prevent collapsing of the tube uponultracentrifugation. The extract was centrifuged at 200,000×g for threehours at 4° C. to sediment membrane vesicles contained in the MS15. Atthe end of centrifugation, the oil was discarded. The supernatant wascarefully collected and placed in a cold 1.5-ml tube on ice. Severalsupernatants were used if necessary. This supernatant is referred to asMS200 (or mitotic cytosolic extract). The MS200 extract was aliquotedand frozen as described for the M515 extract.

Chromatin Transfer On the day before the cloning procedure, one 35 mmNunc was prepared with 1 million fibroblast cells, or six T75 flaskscontaining 500,000 cells were prepared for shake-off. On the morning ofthe cloning procedure, the Alpha MEM with 15% FBS irradiated, completemedia in all flasks was changed to remove debris and dead cells. Priorto performing the cell permeabilization and mitotic extract reaction,the lowest concentration of SLO necessary to permeabilize 80-90% of thecells was determined by serial dilution of the SLO stock solution (e.g.,dilutions of 1×, 0.5×, 0.3×, and 0.1×), incubating for 30 minutes atapproximately 38.5° C. in a water bath, and then staining with propidiumiodide.

The cells were dissociated from confluent culture or newly transfectedcell culture using trypsin or cell dissociation buffer, placed in 15 mlconical tube, and washed once by centrifugation using Hank's BalancedSalt Solution, Ca/Mg free (HBSS). The cell pellet was resuspended in 1ml HBSS, and the cells were counted to determine the concentration.Approximately 50,000-100,000 cells were suspended in 100 μl HBSS (GibcoBRL, cat. No. 14170-120) at room temperature. The 5 μl SLO stocksolution at the previously determined concentration was added. Themixture was incubated at approximately 38.5° C. for 30 minutes in awater bath. The tube was gently tapped 2-3 times during incubation toensure that the cells remained in suspension. A 200 μl volume of roomtemperature PBS (Ca/Mg free) was added and mixed well by gentlepipetting. The cells were centrifuged at 500×g for five minutes at roomtemperature in table top centrifuge. All of the supernatant wasdiscarded. The pellet was small and may not be clearly visible.

A 40 μl volume of mitotic extract containing the ATP-generating systemwas added, and the solution was mixed well. The extract was preparedduring the 30 minute incubation above. One vial of 40 μl extract wasthawed and added to 1.2 μl of ATP-generating system. The solution wasmixed well and kept at room temperature. The mixture was incubated at38.5° C. in a water bath for 30 minutes, and the tube was occasionallygently tapped. A 500 μl volume of room temperature complete media (AlphaMEM+15% Fetal calf serum) was added and stored in an approximately 38.5°C. incubator with the lid open until use. The cells were centrifuged at500×g for five minutes at room temperature in a table top centrifuge.The cell pellet was resuspended in 1 ml room temperature TL Hepes andtransferred to a 15 ml conical tube. A 1 ml volume of 3 mg/ml Proteasein TL Hepes (filtered) was added for a final protease concentration onthe cells of 1.5 mg/ml, and the mixture was incubated for 30 seconds. TLHepes was added to fill the 15 ml conical tube, and the tube was cappedand centrifuged at 2300 rpm for five minutes. TL Hepes was removed fromcells, and 150 ul of TL Hepes was added to cells. The tube was labeledfor the appropriate cell line and placed on a warming stage.

During the time that the cells were being prepared, the manipulationstation was prepared for cell transfer using an appropriately sized celltransfer pipette. Approximately 50 enucleated oocytes were placed into a50 ul drop of TL Hepes under heavy mineral oil in a 100 mm dish. Thewashed cells were placed in a drop with enucleated oocytes. Enough cellswere used for genetic transfer to all oocytes. Care was taken to avoidusing so many cells that they sterically blocked the manipulation tools.One enucleated oocyte was placed on the holder, and one cell was placedin the perivitellin space under the zona, such that the cell wastouching the plasma membrane of the oocyte. To facilitate fusion, thecell was placed adjacent to, or opposite to the polar body. After allenucleated oocytes had cells transferred to them, oocytes were removedfrom the microdrop and placed in a pre-warmed 35 mm dish of HECM Hepesfor fusion.

Results Comparing the results in Table 10 below for donor cells in whichthe membrane was not resealed prior to genetic transfer with the resultsfor donor cells in which the membrane was resealed prior to genetictransfer indicates that increased cloning efficiency may be obtained bynot resealing the permeabilized cell membrane. Additionally, Table 10demonstrates that the use of donor cells in G₁ phase instead ofconfluent cells may result in increased viability of the reconstitutedoocyte and resulting embryo. The data in Table 9 was obtained using anextract from bovine primary fibroblasts and donor bovine fetalfibroblasts. MDBK cells have also been successfully used to generatereprogramming extracts. TABLE 9 Embryo development with HAC cell lines:NT Vs SLOT with resealed donor cells Pregnancy at Treatment Total NoBlast (%) Recips 40 d (%) 60 d (%) 90 d (%) 120 d (%) 150 d (%) HAC NTs8872 1124 (18) 508 170 (34) 89 (18) 82 (16) 76 (15) 72 (14) HAC SLOTs2709  223 (12) 91  42 (46) 22 (24) 19 (21) 18 (20) 17 (19) Total 115811347 (17) 599 212 (35) 111 (19)  101 (17)  94 (16) 89 (15)

TABLE 10 Embryo development with ΔHAC and ΔΔHAC cell lines: confluentdonor cells (CTC) vs G₁ donor cells (CTD) without resealing of donorcell membranes Preg Preg Treatment Total No Blast (%) Recips 40 d (%) 60d (%) ΔHAC CTC 1729 185 (15) 68 14/39 (36) 5/13 (38) ΔHAC CTD 1177 181(22) 68 25/44 (56) 5/22 (33) ΔΔHAC CTC 1230 147 (17) 60 ΔΔHAC CTD 521 95 (26) 29 Total 4657 608 (19) 225

EXAMPLE 12 Methods for the Generation of Chimeric Mammals

Many spontaneous abortions that occur using traditional methods to clonemammals are thought to result from placental abnormalities rather thanfrom problems with the fetus. Thus, methods have been developed toproduce chimeric embryos with placental tissue primarily from one origin(e.g., an in vitro fertilized, naturally-occurring, orparthenogenetically activated embryo) and fetal tissue primarily fromanother origin (e.g., a nuclear transfer embryo encoding a xenogenousantibody). Chimeric embryos with placental tissue derived primarily fromcells from in vitro fertilized, naturally-occurring, orparthenogenetically activated embryos may better resemblenaturally-occurring placental tissue and result in increased productionof viable offspring.

Preferably, the majority of the cells of the offspring are derived fromcells from the nuclear transfer embryo and thus have a genome that issubstantially identical to that of the donor cell used to generate thenuclear transfer embryo.

In one such method, cells from an in vitro fertilized embryo areinjected into the periphery of a compaction embryo encoding a xenogenousantibody (e.g., between the zona pellucida and the embryo itself) thatwas produced using traditional nuclear transfer methods or any of theother cloning methods described herein. In an alternative method, cellsfrom a precompaction, in vitro fertilized embryo are incubated withcells from a precompaction embryo encoding a xenogenous antibodyproduced using one of the cloning methods of the present invention(e.g., using a reprogrammed chromatin mass or a permeabilized cell asthe donor source) under conditions that allow cells from each embryo toreorganize to produce a single chimeric embryo (Wells and Powell,Cloning 2:9-22, 2000). In both methods, the cells from the in vitrofertilized embryo are preferentially incorporated into the placenta, andthe cells from the nuclear transfer method are preferentiallyincorporated into the fetal tissue. These methods are described furtherbelow. These results were generated using nuclear transfer embryos thatdo not contain a xenogenous antibody gene; however, similar results areexpected for nuclear transfer embryos containing a xenogenous antibodygene.

Isolation of G1 Fibroblasts For the isolation of G1 fibroblasts as donorcells to produce nuclear transfer embryos, the previously described“shake off” method was used (Kasinathan et al., Nature biotech.19:1176-1178, 2001). Briefly, 24 hours prior to isolation, 5.0×10⁵ cellswere plated onto 100 mm tissue culture plates containing 10 ml of α-MEMplus FCS. The following day, plates were washed with PBS, and theculture medium was replaced for one to two hours before isolation. Theplates were then shaken for 30-60 seconds on a Vortex-Genie 2 (FisherScientific, Houston, Tex., medium speed). The medium was removed, spunat 500×g for five minutes, and the pellet was re-suspended in 250 μl ofMEM plus FCS. This cell suspension consisted of newly divided celldoublets attached by a cytoplasmic bridge, some single cells, andmetaphase or anaphase cells. The cell doublets attached by a cytoplasmicbridge were used as donor cells for nuclear transfer.

Nuclear Transplantation, Activation, and Embryo Culture The nucleartransfer procedure using the isolated G1 fibroblasts was performedessentially as previously described (Cibelli et al., Nature Biotech.16(7):642-646, 1998; Kasinathan et al., Biol. Reprod. 64(5):1487-1493,2000). In vitro matured oocytes were enucleated about 18-20 hours postmaturation, and chromosome removal was confirmed by bisBenzimide(Hoechst 33342, Sigma) labeling under UV light. These cytoplast-donorcell couplets were fused using a single electrical pulse of 2.4 kV/cmfor 20 microseconds (Electrocell manipulator 200, Genetronics, SanDiego, Calif.). At 30 hours past maturation, reconstructed oocytes andcontrols were activated with calcium ionophore (5 μM) for four minutes(Cal Biochem, San Diego, Calif.) and 10 μg cycloheximide and 2.5 μgcytochalasin D (Sigma) in ACM culture medium (100 mM NaCl, 3 mM KCl,0.27 Mm CaCl₂, 25 mM NaHCO₃, 1 mM sodium lactate, 0.4 mM Pyruvate, 1 mML-glutamine, 3 mg/ml BSA (fatty acid free), 1% BME amino acids, and 1%MEM nonessential amino acids; all from Sigma) for six hours as describedpreviously (Liu et al., Mol. Reprod. Dev. 49:298-307, 1998; Presicce etal., Mol. Reprod. Dev. 38:380-385, 1994). After activation, eggs werewashed in HEPES buffered hamster embryo culture medium (HECM-HEPES, 114mM NaCl, 3.2 mM KCl, 2 mM CaCl₂, 10 mM Sodium Lactate, 0.1 mM sodiumpyruvate, 2 mM NaHCO₃, 10 mM HEPES, and 1% BME amino acids; Sigma) fivetimes and placed in culture in 4-well tissue culture plates containingmouse fetal fibroblasts and 0.5 ml of embryo culture medium covered with0.2 ml of embryo tested mineral oil (Sigma). Twenty five to 50 embryoswere placed in each well and incubated at 38.5° C. in a 5% CO₂ in airatmosphere. On day four, 10% FCS was added to the culture medium. Ondays seven and eight, development to the blastocyst stage was recorded.

Bovine In vitro Fertilization In vitro fertilization was performed asdescribed earlier to produce bovine in vitro fertilized embryos (Collaset al., Mol. Reprod. Dev. 34:224-231, 1993). A 45% and 90% isotonicPercoll gradient was prepared with sperm TL stock (Parrish et al.,Theriogenology 24:537-549, 1985). Frozen-thawed bovine sperm from asingle bull was layered on top of the gradient and centrifuged for 30minutes at 700×g (2000 rpm using a 6.37 inch tip radius). Theconcentration of sperm in the pellet was determined, and the sperm wasdiluted in sperm TL (sperm TL stock, 1 mM pyruvate, 6 mg/ml BSA, and 1%PS) such that the final concentration at fertilization was 10⁶ sperm/ml.At 22 hours post maturation, oocytes were wash three times in TL HEPESand placed in 480 ul of fertilization TL (Bavister et al., Biol. Reprod.28:235-247, 1983) in Nunc wells containing 6 mg/ml BSA, 0.2 mM pyruvate,20 uM penicillamine, 10 uM hypotaurine, 1 mM epinephrine (Leibfried etal., J. Reprod. Fertil. 66:87-93, 1982), and 0.004 ug/ml heparin. Twentymicroliters of sperm were added to generate a final concentration of 10⁶sperm/ml to 50 oocytes. Culture conditions were the same as thosedescribed above for nuclear transfer. Fertilization rates were over 90%based on pronuclear development.

Chimeric Nuclear Transfer Embryos In vitro fertilized embryos at 8-cellstage (6-12 blastomeres) were harvested at approximately 96 hours postfertilization, prior to compaction. The zona pellucida was removed withprotease (3 mg/ml in TL-HEPES). The zona dissolution was carefullymonitored using a dissecting microscope. When the zona first appeared todissolve (˜two minutes), the embryos were removed and washed in TL-HEPESand transferred to 30 mm petri dishes containing Hank's balanced saltsolution and incubated at 37.5° C. for 30 minutes. The blastomeres fromthese precompaction embryos were transferred into microdrops (50 μl) ofTL-HEPES under mineral oil in 100 mm petridish. Nuclear transfer embryoson day four at the 8-16 cell stage were selected and transferred intothe same microdrops containing the blastomeres. These nuclear transferembryos included both precompaction embryos (e.g., 8 cell stage embryos)and compaction embryos (e.g., 16 stage embryos). Then 4-6 blastomereswere transferred into the nuclear transfer embryos with the beveledmicro pipette (35 μm diameter) using standard micromanipulationtechniques. After transferring the blastomeres, the embryos werecultured as described for nuclear transfer embryos.

On days seven and eight, the development to blastocyst of the chimericembryos was evaluated. The blastocysts were also analyzed for thepresence of the membrane dye DiI that was added to the cells from the invitro fertilized embryo before they were injected into the nucleartransfer embryo. The cells were labeled on day four and observed on dayseven. This dye is maintained for a few cell divisions in the progeny ofthe originally dyed cells, allowing the chimeric embryo to be analyzedafter a few cell divisions. Based on this analysis, cells from the invitro fertilized embryo were incorporated into the chimeric embryo. Ifdesired, fluorescence in situ hybridization (FISH) with a probe specificfor a nucleic acid in either the in vitro fertilized embryo or thenuclear transfer embryo can be performed using standard methods (see,for example, Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, New York, pp. 14.7.1-14.7.12, 1995). This FISHanalysis can be used to determine the distribution of cells derived fromeach embryo in the chimeric embryo (e.g., to determine what percent ofthe cells are incorporated into the inner cell mass and what percent areincorporated into the trophectoderm) while it is cultured in vitro andin the fetus or the offspring generated from the embryo. Alternatively,a reporter gene such as green fluorescent protein can be added to cellsfrom one of the embryos and used to monitor the incorporation of thecells into the placenta and various fetal tissues of the chimericembryo.

Embryo Transfer Days seven and eight, nuclear transfer blastocysts ofgrade 1 and 2, derived from nuclear transfer embryos and chimericnuclear transfer embryos were transferred into day six and sevensynchronized recipient heifers. Recipients were synchronized using asingle injection of Lutalyse (Parmacia & Upjohn, Kalamazoo, Mich.)followed by estrus detection. The recipients were examined on days 30and 60 after embryo transfer by ultrasonography for the presence ofconceptus and thereafter every 30 days by rectal palpation until 240days. The pregnancy results at day 40 for the chimeric embryos and forcontrol embryos produced by fusing a transgenic bovine fibroblast withan oocyte are compared in Table 11. These results indicate that agreater number of chimeric embryos survived until day 40. TABLE 11Embryo transfers and pregnancies Control Nuclear transfers ChimericNuclear Transfers 40 day 40 day Implant No of recipients Pregnancy No ofrecipients Pregnancy First 2 1 2 1 Second 6 1 4 3 Total 8 2 (25%) 6 4(67%)

Alternative Methods for Production of Chimeric Embryos Standard methodscan be used to modify the above method for producing chimeric embryos.For example, a naturally-occurring embryo can be surgically isolatedfrom a mammal (e.g., a bovine) or an oocyte can be parthenogeneticallyactivated using standard techniques and used instead of the in vitrofertilized embryo. If desired, fewer cells from the in vitro fertilized,naturally-occurring, or parthenogenetically activated embryos (e.g., 1,2, 3, 4, or 5 cells) can be injected into the nuclear transfer embryo toreduce the percent of the injected cells and their progeny that becomeincorporated into fetal tissue. Alternatively, more cells (e.g., 6, 7,8, 9, 10, 11 or more cells) can be injected to increase the percent ofthe injected cells and their progeny that are incorporated intoplacental tissue. Moreover, cells from embryos in other cell stages canbe used. For example, in vitro fertilized, naturally-occurring, orparthenogenetically activated embryos at the 4, 8, 16, 32, 64, 128, 256,512, or later cell stage can be injected into nuclear transfer embryosat the 4, 8, 16, 32, 64, 128, 256, 512, or later cell stage. Theinjected cells and the nuclear transfer embryo can be at the same cellstage or at different cell stages. In one embodiment, the in vitrofertilized, naturally-occurring, or parthenogenetically activated embryohas increased ploidy (e.g., a DNA content of 4n) relative to the nucleartransfer embryo, which further biases the injected cells to thetrophectoderm (i.e., the outermost layer of cells of the embryo thatprimarily forms the placental tissue). If desired, all or part of thezona pellucida can be kept surrounding the injected cells, rather thanremoved prior to injection.

In other alternative methods, cells from a precompaction or compactionin vitro fertilized, naturally-occurring, or parthenote embryo areincubated with cells from a precompaction nuclear transfer embryo underconditions that allow cells from each embryo to reorganize to produce asingle chimeric embryo (Wells and Powell, Cloning 2:9-22, 2000). Cellsfrom in vitro fertilized, naturally-occurring, or parthenote embryo areexpected to contribute primarily to the trophectoderm and eventually tothe placental tissue, and cells from the nuclear transfer embryo areexpected to contribute primarily to the inner cell mass and eventuallyto the fetal tissue. Cells from both embryos can be at the same cellstage or at different cell stages, and the same or different numbers ofcells from each embryo can be combined to form the aggregation embryo.

If desired, a cell from the resulting cloned fetus or the clonedoffspring can be used in a second round of nuclear transfer to generateadditional cloned offspring. Cells from the initial cloned fetus orcloned offspring may also be frozen to form a cell line to be used as asource of donor cells for the generation of additional cloned ungulates.

Optional Elimination of Cells from Non-Transgenic Embryo

If desired, to reduce further the number of cells and their progeny fromthe an in vitro fertilized, naturally-occurring, or parthenogeneticallyactivated embryo that are incorporated into the fetal tissue oroffspring, an antibody that is reactive with an antigen (e.g., a B-cellor germ cell antigen, a cell-surface antigen, or any antigen present inor on cells from the fertilized, naturally-occurring, orparthenogenetically activated embryo but not present in or on cells fromthe nuclear transfer embryo) from the in vitro fertilized,naturally-occurring, or parthenogenetically activated embryo isadministered to the chimeric embryo, fetus, or offspring in an amountsufficient to reduce the quantity and/or activity of cells from the invitro fertilized, naturally-occurring, or parthenogenetically activatedembryo that are incorporated into the fetus or offspring. In preferredembodiments, between 1 and 10 mg, 10 and 25 mg, 25 and 50 mg, 10 and 100mg, 50 and 100 mg, or 100 to 500 mg of the antibody is administered inone or multiple doses to the fetus. Preferably, at least 0.25, 0.5, 1.0,1.5, or 2 grams of the antibody is administered in one or multiple dosesto the offspring. Preferably, the antibody is administered prior tocolostrum.

In another method for generating chimeric fetuses or offspring, cellsfrom one of the initial embryos used to produce the chimeric fetus oroffspring have a nucleic acid encoding a xenogenous antibody (e.g., ahuman antibody). Additionally, cells from the aforementioned initialembryo or another initial embryo have a nucleic acid encoding anantibody that is reactive with an endogenous antibody (e.g., an antibodynaturally produced by cells from any of the initial embryos used togenerate the chimeric fetus or offspring) and that reduces the amount oractivity of an endogenous antibody in the resulting fetus or offspring.

The nucleic acid encoding the antibody reactive with an endogenousantibody can be obtained using standard molecular biology techniques.For example, an mRNA from a B-cell producing an antibody reactive withungulate antibodies can be reverse-transcribed, and the resulting cDNAcan be inserted into the donor cell, nucleus, or chromatin mass used toform one of the initial embryos. In some embodiments, the cDNA isinserted into the HAC containing a xenogenous immunoglobulin nucleicacid, and the HAC is inserted into the donor cell, nucleus, or chromatinmass using the methods described herein. If desired, the cDNA can beplaced under the control of a cell-specific promoter, such as aliver-specific promoter.

In one such method, a cell, nucleus, or chromatin mass is inserted intoan oocyte, thereby forming a first embryo. The cell, nucleus, orchromatin mass has a nucleic acid encoding a xenogenous first antibodyand a nucleic acid encoding a second antibody reactive with anendogenous antibody. One or more cells from the first embryo arecontacted with one or more cells from a second embryo (e.g., an in vitrofertilized embryo, naturally-occurring embryo, or parthenogeneticallyactivated embryo), thereby forming a third embryo. The third embryo istransferred into the uterus of a host mammal under conditions that allowthe third embryo to develop into a fetus or live offspring. Theresulting fetus or offspring expresses the xenogenous first antibody andthe second antibody, and the second antibody reduces the quantity and/oractivity of an endogenous antibody.

In a related method, a permeabilized cell is incubated in areprogramming media under conditions that allow the removal of a factorfrom a nucleus, chromatin mass, or chromosome of the permeabilized cellor the addition of a factor from the reprogramming media to the nucleus,chromatin mass, or chromosome, thereby forming a reprogrammed cell. Thecell has a nucleic acid encoding a xenogenous first antibody and anucleic acid encoding a second antibody reactive with an endogenousantibody. The reprogrammed cell is inserted into an oocyte, therebyforming a first embryo. One or more cells from the first embryo arecontacted with one or more cells from a second embryo (e.g., an in vitrofertilized embryo, naturally-occurring embryo, or parthenogeneticallyactivated embryo), thereby forming a third embryo. The third embryo istransferred into the uterus of a host mammal under conditions that allowthe third embryo to develop into a fetus or live offspring. Theresulting fetus or offspring expresses the xenogenous first antibody andthe second antibody, and the second antibody reduces the quantity and/oractivity of an endogenous antibody.

EXAMPLE 13 Transgenic Ungulates Producing Xenogenous Antibodies thathave a Mutation in One or More Endogenous Antibodies

The expression of endogenous antibodies may be further reduced bymutating one or more endogenous antibody genes. By increasing the numberof functional xenogenous immunoglobulin heavy or light chain genesrelative to the number of functional endogenous heavy or light chaingenes, the percentage of B-cells expressing xenogenous antibodies shouldincrease. If desired, an antibody may be administered to eliminate theresidual endogenous B-cells and antibodies as described below.

To generate these transgenic ungulates, ΔHAC or ΔΔHAC transgenicungulates may be mated with transgenic ungulates containing a mutationin one or both alleles of an endogenous immunoglobulin chain (e.g., a muheavy chain or a lambda or kappa light chain). If desired, the resultingtransgenic ungulates may be mated with (i) transgenic ungulatescontaining a mutation in one or both alleles of an endogenousalpha-(1,3)-galactosyltransferase, prion, and/or J chain nucleic acid or(ii) transgenic ungulates containing an exogenous J chain nucleic acid(e.g., human J chain). Alternatively, a cell (e.g., a fetal fibroblast)from a ΔHAC or ΔΔHAC transgenic fetus may be genetically modified by themutation of one or more endogenous immunoglobulin genes. In anotherpossible method, ΔHAC or ΔΔHAC is introduced into a cell (e.g., a fetalfibroblast) in which endogenous immunoglobulins (mu heavy and/or lambdalight chains) are hemizygously or homozygously inactivated. In any ofthe above methods, the cells may also be genetically modified by (i) theintroduction of a mutation, preferably a knockout mutation, into one orboth alleles of an endogenous alpha-(1,3)-galactosyltransferase, prion,and/or J chain nucleic acid or (ii) the introduction of an exogenous Jchain nucleic acid. The resulting transgenic cell may then be used innuclear transfer procedures to generate the desired transgenicungulates. Exemplary methods are described below.

DNA Constructs The mu heavy chain (FIG. 2A), lambda light chain, kappalight chain, alpha-(1,3)-galactosyltransferase, prion, and/or J chainknockout constructs described above may be used. Alternatively, thepuromycin resistant mu heavy chain construct described below may be used(FIG. 3F). This knockout construct was designed to remove the 4 maincoding exons of the bovine mu heavy chain locus but leave thetransmembrane domain intact, resulting in the inactivation of the muheavy chain locus.

The puromycin resistant construct was assembled as follows. A 4.4kilobase XhoI fragment containing the region immediately proximal tocoding exon 1 was inserted into the XhoI site of pBluescript II SK+.Plasmid pPGKPuro, which contains a puromycin resistant gene, wasobtained from Dr. Peter W. Laird, Whitehead Institute, USA. A 1.7 KbXhoI fragment containing a puromycin resistance gene was subclonedadjacent to, and downstream of, the 4.4 Kb fragment into the SalI sitepresent in the polylinker region. This 1.7 Kb puromycin marker replacesthe coding exons CH1, CH2, CH3 and CH4 of the bovine immunoglobulinheavy chain locus. An XbaI fragment containing a 4.6 Kb region of the mulocus that is downstream of these four exons in the wild-type genomicsequence was added to this construct for use as the second region ofhomology.

To generate the final targeting construct, a subclone of this constructwas generated by cutting the three assembled fragments with NotI andMluI The MluI restriction digestion truncates the 4.6 Kb fragment downto 1.4 Kb. The NotI site lies in the polylinker and does not cut intothe subcloned DNA itself. The MluI site was filled in with a Klenowfragment to generate a blunt end, and the NotI/filled in MluI fragmentwas subcloned into a fresh pBluescript II SK+ vector using the NotI andSmaI sites present in the pBluescript vector. For gene targeting, thefinal vector is linearized with NotI.

Gene Targeting by Electroporation and Drug Selection of TransfectedFibroblasts For electroporation, a single cell suspension of 1×10⁷bovine fetal fibroblasts (e.g, fibroblasts obtained as described inExample 1 from a ΔHAC or ΔΔHAC transgenic fetus) that had undergone alimited number of population doublings is centrifuged at 1200 rpm forfive minutes and re-suspended in 0.8 ml of serum-free Alpha-MEM medium.The re-suspended cells are transferred to a 0.4 cm electroporationcuvette (Invitrogen, cat #.P460-50). Next, 30 μg of a restrictionenzyme-linearized, gene targeting vector DNA is added, and the contentsof the cuvette are mixed using a 1 ml pipette, followed by a two minuteincubation step at room temperature. The cuvette is inserted into theshocking chamber of a Gene Pulser II electroporation system (Biorad) andthen electroporated at 1000 volts and 50 μF. The cuvette is quicklytransferred to a tissue culture hood and the electroporated cells arepipetted into approximately 30 ml of complete fibroblast medium. Thecells are equally distributed into thirty 100 mm tissue culture dishes(Corning, cat #.431079), gently swirled to evenly distribute the cells,and incubated at 38.5° C./5% CO₂ for 16 to 24 hours The media is removedby aspiration and replaced with complete fibroblast medium containingthe selection drug of choice. The media is changed every two days andcontinued for a total time period of 7 to 14 days. During the drugselection process, representative plates are visually monitored to checkfor cell death and colony formation. Negative control plates are set upthat contained fibroblasts that are electroporated in the absence of thegene targeting vector and should yield no colonies during the drugselection process.

Picking of Drug Resistant Fibroblast Colonies and Expansion of CellsFollowing completion of the drug selection step (usually 7 to 14 days),the drug resistant colonies are macroscopically visible and ready fortransfer to 48 well tissue culture plates for expansion. To assist inthe transferring process, individual colonies are circled on the bottomof the tissue culture plate using a colored marker (Sharpie). Tissueculture plates containing colonies are washed 2× with 1×D-PBS (withoutCa²⁺ and Mg²⁺) and then 5 ml of a 1:5 dilution of the cell dissociationbuffer is added per plates. Following a 3 to five minute roomtemperature incubation step, individual colonies start to detach fromthe bottom of the tissue culture dish. Before the colonies detached,they are individually transferred to a single well of a 48 well tissueculture plate using a P200 pipetmen and an aerosol barrier pipette tip(200 or 250 μl). Following transfer, the colony is completelydissociated by pipetting up-and-down and 1 ml of complete fibroblastmedium is added. To ensure that the cells are drug resistant, drugselection is continued throughout the 48 well stage. The transferredcolonies are cultured at 38.5° C./5% CO₂ and visually monitored using aninverted microscope. Two to seven days later, wells that are approachingconfluency are washed two times with 1×D-PBS (without Ca²⁺ and Mg²⁺) anddetached from the bottom of the well by the addition of 0.2 ml of celldissociation buffer, followed by a five minutes room temperatureincubation step. Following detachment, the cells are further dissociatedby pipetting up-and-down using a P1000 pipetmen and an aerosol pipettetip (1000 μl). Approximately 75% of the dissociated fibroblasts aretransferred to an individual well of a 24 well tissue culture plate toexpand further for subsequent PCR analysis and the remaining 25% istransferred to a single well of a second 24 well plate for expansion andeventually used for somatic cell nuclear transfer experiments. Whencells in the plate containing 75% of the original cells expanded to nearconfluency, DNA is isolated from that clone for genetic analysis.

DNA Preparation The procedure used to isolate DNA for genetic analysesis adapted from Laird et al, Nucleic Acids Research, 1991, Volume 19,No. 15. In particular, once a particular clone has attainednear-confluency in one well of a 24 well plate, culture medium isaspirated from that well and the adherent cells are washed twice withPBS. The PBS is aspirated off and replaced with 0.2 ml buffer to lysethe cells and digest excess protein from the DNA to be isolated. Thisbuffer is composed of 100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 0.2% SDS, 200mM NaCl and 100 ug/ml proteinase K. The 24 well plate is returned to thetissue culture incubator for a minimum of three hours to allow therelease of the DNA and digestion of protein. The viscous product of thisprocedure is transferred to a 1.5 ml microcentrifuge tube and 0.2 ml ofisopropanol added to precipitate the DNA. The precipitate is recoveredby centrifugation, the DNA pellet is rinsed with 70% ethanol, and afterair-drying, the pellet is resuspended in 25-50 ul of buffer containing10 mM Tris, pH 8, and 1 mM EDTA. This DNA is used for PCR analyses ofclones.

Screening of Clones Two different approaches are used to screen clones,both employing the polymerase chain reaction (PCR). All approachesdescribed in this section are adaptable to the targeting of any othergene, the only difference being the sequences of the primers used forgenetic analysis.

According to the first approach, two separate pairs of primers are usedto independently amplify products of stable transfection. One pair ofprimers is used to detect the presence of the targeting vector in thegenome of a clone, regardless of the site of integration. The primersare designed to anneal to DNA sequences both present in the targetingvector. The intensity of the PCR product from this PCR reaction may becorrelated with the number of copies of the targeting vector that haveintegrated into the genome. Thus, cells containing only one copy of thetargeting vector tend to result in less intense bands from the PCRreaction. The other pair of primers is designed to detect only thosecopies of the vector that integrated at the desired locus. In this case,one primer is designed to anneal within the targeting vector and theother is designed to anneal to sequences specific to the locus beingtargeted, which are not present in the targeting vector. In this case, aPCR product is only detected if the targeting vector has integrateddirectly next to the site not present in the targeting vector,indicating a desired targeting event. If product is detected, the cloneis used for nuclear transfer.

For the neomycin resistant heavy chain knockout construct, primers Neo1(5′-CTT GAA GAC GAA AGG GCC TCG TGA TAC GCC-3′, SEQ ID NO: 42) andIN2521 (5′-CTG AGA CTT CCT TTC ACC CTC CAG GCA CCG-3′, SEQ ID NO: 43)are used to detect the presence of the targeting vector in cells,regardless of the location of integration. Primers Neo1 and OUT3570(5′-CGA TGA ATG CCC CAT TTC ACC CAA GTC TGT C-3′, SEQ ID NO: 44) areused to specifically amplify only those copies of the targetingconstruct that integrated into the mu heavy chain locus.

For these PCR reactions to analyze the integration of the neomycinresistant heavy chain knockout construct, a Qiagen PCR kit is used. ThePCR reaction mixture contains 1 pmole of each primer, 5 ul of 10×reaction buffer, 10 μl of Q solution, 5 μl of DNA, and 1 μl of dNTPsolution. The reaction mixture is brought to a total volume of 50 ulwith H₂O. This PCR amplification is performed using an initialdenaturing incubation at 94° C. for two minutes. Then, 30 cycles ofdenaturation, annealing, and amplification are performed by incubationat 94° C. for 45 seconds, 60° C. for 45 seconds, and 72° C. for twominutes. Then, the reaction mixture is incubated at 72° C. for fiveminutes and at 4° C. until the mixture is removed from the PCR machine.

In the alternative approach, a single primer set is used to amplify thetargeted locus and the size of the PCR products is diagnostic forcorrect targeting. One primer is designed to anneal to a region of thelocus not present in the targeting vector and the other primer isdesigned to anneal to a site present in the targeting vector but alsopresent in the wild type locus. In this case, there is no detection oftargeting vector that had integrated at undesirable sites in the genome.Because the region deleted by the targeting vector is different in sizefrom the drug selection marker inserted in its place, the size of theproduct depended on whether the locus amplified is of wild-type genotypeor of targeted genotype. Amplification of DNA from clones containingincorrect insertions or no insertions at all of the targeting vectorresults in a single PCR product of expected size for the wild typelocus. Amplification of DNA from clones containing a correctly targeted(“knocked out”) allele results in two PCR products, one representingamplification of the wild type allele and one of altered, predictablesize due to the replacement of some sequence in the wild-type allelewith the drug resistance marker, which is of different length from thesequence it replaced.

For the puromycin resistant heavy chain knockout construct, primersShortend (5′-CTG AGC CAA GCA GTG GCC CCG AG-3′, SEQ ID NO: 45) andLongend (5′-GGG CTG AGA CTG GGT GAA CAG AAG GG-3′, SEQ ID NO: 46) areused. This pair of primers amplifies both the wild-type heavy chainlocus and loci that have been appropriately targeted by the puromycinconstruct. The size difference between the two bands is approximately0.7 Kb. The presence of the shorter band is indicative of appropriatetargeting.

For this PCR reaction to analyze the integration of the puromycingresistant heavy chain knockout construct, a Promega Master Mix kit isused. The PCR reaction mixture contains 1 pmole of each primer, 2.5 μlof DNA, and 25 μl of 2× Promega Master Mix. The reaction mixture isbrought to a total volume of 50 μl with H₂O. This PCR amplification isperformed using an initial denaturing incubation at 94° C. for twominutes. Then, 30 cycles of denaturation, annealing, and amplificationare performed by incubation at 94° C. for 45 seconds, 60° C. for 45seconds, and 72° C. for two minutes. Then, the reaction mixture isincubated at 72° C. for five minutes and at 4° C. until the mixture isremoved from the PCR machine.

First Round of Nuclear Transfer Selected fibroblast cells in which animmunoglobulin gene has been inactivated may be used for nucleartransfer as described in Example 1 to generate a transgenic ungulatecontaining a mutation in an endogenous immunoglobulin gene andcontaining a HAC encoding a xenogenous immunoglobulin gene.Alternatively, nuclear transfer may be performed using standard methodsto insert a nucleus or chromatin mass (i.e., one or more chromosomes notenclosed by a membrane) from a selected transgenic fibroblast into anenucleated oocyte (U.S. Ser. No. 60/258,151; filed Dec. 22, 2000). Thesemethods may also be used for cells in which an endogenousalpha-(1,3)-galactosyltransferase, prion, and/or J chain nucleic acidhas been mutated.

Second Round of Mutagenesis and Nuclear Transfer If desired, a cell(e.g., a fetal fibroblast) may be obtained from a transgenic ungulategenerated from the first round of nuclear transfer. Another round ofgene targeting may be performed as described above to inactivate thesecond allele of the gene inactivated in the first round of targeting.Alternatively, another immunoglobulin (e.g., mu heavy chain, lambdalight chain, kappa light chain, or J chain),alpha-(1,3)-galactosyltransferase, or prion gene may be inactivated inthis round of targeting. For this second round of targeting, either ahigher concentration of antibiotic may be used or a knockout constructwith a different antibiotic resistance marker may be used. Antibioticresistance cells may be selected as described above. The selected cellsmay be used in a second round of nuclear transfer as described above togenerate, for example, a transgenic ungulate containing two mutations inendogenous immunoglobulin genes and containing a HAC encoding axenogenous immunoglobulin gene. Alternatively, the selected antibioticresistant cells may first be treated to isolate G1 phase cells asdescribed below, which are used for the second round of nucleartransfer.

For isolation of G1 cells for nuclear transfer, 5.0×10⁵ cells are platedonto 100 mm tissue culture plates containing 10 ml of α-MEM+FCS, twentyfour hours prior to isolation. The following day, plates are washed withPBS and the culture medium is replaced for 1-2 hours before isolation.The plates are then shaken for 30-60 seconds on a Vortex-Genie 2 (FisherScientific, Houston, Tex., medium speed), the medium is removed, spun at1000 G for five minutes and the pellet is re-suspended in 250 μl ofMEM+FCS. Newly divided cell doublets attached by a cytoplasmic bridge,are then selected, as these cells are in early G1. This isolationprocedure is referred to as the “shake off” method.

EXAMPLE 14 Additional Methods to Mutate Endogenous Immunoglobulin Genes

In some embodiments of the present approach, xenogenous immunoglobulinproduction is accomplished essentially by the combined use of homologousrecombination techniques, introduction of artificial chromosomescarrying entire xenogenous Ig loci, nuclear transfer, and administrationof an antibody to eliminate endogenous antibody. More specifically, theprocess preferably involves the targeted disruption of one or bothalleles of the IgM heavy chain gene, and optionally one or both allelesof the Ig light chain gene, although xenogenous antibody production canalso be accomplished in wild-type animals (i.e., animals without Igknock outs). Gene knock outs may be effected by sequential homologousrecombination, then another mating procedure. In a preferred embodiment,this is effected by initially effecting targeted disruption of oneallele of the IgM heavy chain gene of a male or female ungulate (forexample, bovine) fetal fibroblast in tissue culture using a suitablehomologous recombination vector. The use of fetal fibroblasts ispreferred over some other somatic cells as these cells are readilypropagated and genetically manipulated in tissue culture. However, theuse of fetal fibroblasts is not essential to the invention, and indeedother cell lines may be substituted therefor with equivalent results.

This process, of course, entails constructing a DNA construct havingregions of homology to the targeted IgM heavy chain allele such that theconstruct upon integration into an IgM heavy chain allele in theungulate genome disrupts the expression thereof. An exemplary vector forcarrying out such targeted disruption of an IgM allele is described inthe example which follows. In this regard, methods for constructingvectors that provide for homologous recombination at a targeted site arewell known to those skilled in the art. Moreover, in the presentinstance, the construction of a suitable vector is within the level ofskill in the art, given especially that the sequence of the bovine IgMheavy chain and Ig lambda light chain genes are known, as are thesequences of immunoglobulin genes from other ungulates (see below) Inorder to facilitate homologous recombination, the vectors used to effecthomologous recombination and inactivation of the IgM gene, respectively,comprise portions of DNA that exhibit substantial sequence identity tothe ungulate IgM heavy and Ig light chain genes. Preferably, thesesequences possessing at least 98% sequence identity, more preferably, atleast 99% sequence identity, and still more preferably will be isogenicwith the targeted gene loci to facilitate homologous recombination andtargeted deletion or inactivation.

Typically, and preferably the construct will comprise a marker gene thatprovides for selection of desired homologous recombinants, for example,fibroblast cells, wherein the IgM heavy chain gene and/or Ig light chaingene has been effectively disrupted. Exemplary marker genes includeantibiotic resistance markers, drug resistance markers, and greenfluorescent protein, among others. A preferred construct is shown inFIG. 2A and starting materials used to make this construct in FIGS. 3Aand 3B. Other constructs containing two regions of homology to anendogenous immunoglobulin gene, which flank a positive selection marker(e.g., an antibiotic resistance gene) that is operably linked to apromoter, may be generated using standard molecular biology techniquesand used in the methods of the present invention.

The mu knockout construct shown in FIGS. 2A and 3C was designed toremove the exons encoding the bovine immunoglobulin heavy chain constantregion, designated as “C-mu exons 1-4” and the two exons encoding thetransmembrane domain, designated “TM exons”.

To construct this vector, the region designated as “1”, an Xba1-Xho1fragment from the genomic mu heavy chain bovine sequence, was subclonedinto the commercial DNA vector, pBluescript (Stratagene, La Jolla,Calif.), previously cut with the enzymes XbaI and XhoI. Once thisfragment was cloned, there was a NotI restriction enzyme recognitionsequence adjacent to the Xba1 site, used to insert a NotI fragment ofapproximately 3.5 Kb. This fragment contains a neomycin resistancemarker, described further below. If desired, other mu knock outconstructs may be constructed using the genomic mu heavy chain sequencefrom another ungulate breed, species, or genus (e.g., the mu heavy chainsequence deposited as Genbank accession number U63637 from a SwissBull/Holstein cross).

Once fragment “1” and the neomycin resistance marker were joinedtogether into pBluescript, there remained a Sac1 site adjacent to theneomycin resistance marker. The new construct was linearized with Sac1and converted to a blunt end by filling in the sticky ends left from theSac1 digest, using DNA polymerase.

The fragment designated “2” was isolated as an XhoI-BstI1071 fragmentand converted to a blunt-ended fragment by filling in the sticky endsleft from the Xho1 and BstI1071 enzymes, using DNA polymerase.

Once finished, the final construct contained region 2, the neomycinresistance marker and region 1, respectively.

For transfection of bovine fibroblasts, the construct was digested withthe restriction enzyme, Kpn1 (two Kpn1 sites are shown in the diagram)and the DNA fragment was used for homologous recombination.

The neomycin resistance construct was assembled as follows. A constructdesignated “pSTneoB” (Katoh et al., Cell Struct. Funct. 12:575, 1987;Japanese Collection of Research Biologicals (JCRB) deposit number:VE039) was designed to contain a neomycin resistance gene under thecontrol of an SV40 promoter and TK enhancer upstream of the codingregion. Downstream of the coding region is an SV40 terminator sequence.The neo cassette was excised from “pSTneoB” as a XhoI fragment. Afterthe ends of the fragment were converted to blunt ends using standardmolecular biology techniques, the blunt ended fragment was cloned intothe EcoRV site in the vector, pBS246 (Gibco/Life Technologies). Thissite is flanked by loxP sites. The new construct, designated“pLoxP-STNeoR”, was used to generate the mu knockout DNA construct. Thedesired fragment of this construct is flanked by loxP sites and NotIsites, which were originally present in the pBS246 cloning vector. Thedesired NotI fragment, which contains loxP-neo-loxP, was used forreplacement of the immunoglobulin mu constant region exons. The SV40promoter operably linked to the neomycin resistance gene activates thetranscription of the neomycin resistance gene, allowing cells in whichthe desired NotI fragment has replaced the mu constant region exons tobe selected based on their resulting antibiotic resistance.

After a cell line is obtained in which the IgM heavy chain allele hasbeen effectively disrupted, it is used as a nuclear transfer donor toproduce a cloned ungulate fetus (for example, a cloned bovine fetus) andeventually a fetus or animal wherein one of the IgM heavy alleles isdisrupted. Thereafter, a second round of gene targeted disruption can beeffected using somatic cells derived therefrom, e.g., fibroblasts, inorder to produce cells in which the second IgM heavy chain allele isinactivated, using a similar vector, but containing a differentselectable marker.

Preferably, concurrent to the first targeted gene disruption, a secondungulate (for example, bovine) somatic cell line is also geneticallymodified, which similarly may be of male or female origin. If the firstcell line manipulated is male, it is preferable to modify a female cellline; vice versa if the first cell line manipulated is female, it ispreferable to select a male cell line. Again, preferably, themanipulated cells comprise ungulate (for example, bovine) fetalfibroblasts.

In a preferred embodiment, the female fetal fibroblast is geneticallymodified so as to introduce a targeted disruption of one allele of theIg lambda light chain gene. This method similarly is carried out using avector having regions of homology to the ungulate (for example, bovine)Ig lambda light chain, and a selectable marker, which DNA construct isdesigned such that upon integration and homologous recombination withthe endogenous Ig light chain results in disruption (inactivation) ofthe targeted Ig lambda light gene.

Once a female fibroblast cell line is selected having the desiredtargeted disruption, it similarly is utilized as a donor cell fornuclear transfer or the DNA from such cell line is used as a donor fornuclear transfer.

Alternatively, this cell may be subjected to a second round ofhomologous recombination to inactivate the second Ig lambda light chainusing a similar DNA construct to that used to disrupt the first allele,but containing a different selectable marker.

Methods for effecting nuclear transfer, and particularly for theproduction of cloned bovines and cloned transgenic bovines have beenreported and are described in U.S. Pat. No. 5,945,577 issued to Stice etal. and assigned to University of Massachusetts. Still, alternativelythe nuclear transfer techniques disclosed in WO 95/16670; WO 96/07732;WO 97/07669; or WO 97/07668, (collectively, Roslin Methods) may be used.The Roslin methods differ from the University of Massachusettstechniques in that they use quiescent rather than proliferating donorcells. All of these patents are incorporated by reference herein intheir entirety. These nuclear transfer procedures will produce atransgenic cloned fetus which can be used to produce a cloned transgenicbovine offspring, for example, an offspring which comprises a targeteddisruption of at least one allele of the Ig light chain gene and/or IgMgene. After such cell lines have been created, they can be utilized toproduce a male and female heavy and light chain hemizygous knockout (Mand F Hemi H/L) fetus and offspring. Moreover, these techniques are notlimited to use for the production of transgenic bovines; the abovetechniques may be used for nuclear transfer of other ungulates as well.

Following nuclear transfer, production of desired animals may beaffected either by mating the ungulates or by secondary gene targetingusing the homologous targeting vector previously described.

As noted previously, a further object of the invention involves creatingmale and female heavy and light chain hemizygous knockouts wherein suchhemizygous knockouts are produced using the cell lines alreadydescribed. This may be affected either by mating of the offspringproduced according to the above described methods, wherein an offspringwhich comprises a disrupted allele of the IgM heavy chain gene is matedwith another offspring which comprises a disrupted allele of the Iglight chain. Alternatively, this may be affected by secondary genetargeting by manipulating a cell which is obtained from an offspringproduced according to the above-described procedures. This will compriseeffecting by homologous recombination targeted disruption of an alleleof the IgM heavy chain gene or allele of the Ig light chain. After acell line is produced which comprises a male and female heavy and lightchain hemizygous knockout (M and F Hemi H/L) it will be used to producea fetus or calf which comprises such a knockout. As noted, this iseffected either by mating or secondary gene targeting.

Once the male and female heavy and light chain hemizygous knockouts areobtained, cells from these animals may be utilized to create homozygousknockout (Homo H/L) fetuses. Again, this is affected either bysequential gene targeting or mating. Essentially, if affected by mating,this will involve mating the male heavy and light chain hemizygousknockout with a female heavy and light chain hemizygous knockout andselection of an offspring which comprises a homozygous knockout.Alternatively, the cells from the hemizygous knockout described abovemay be manipulated in tissue culture, so as to knock out the otherallele of the IgM or Ig light chain (lambda) gene. Secondary genetargeting may be preferred to mating as this may provide for more rapidresults, especially given that the gestation period of ungulates, suchas bovines, is relatively long.

Knockout Procedures to Produce Transgenic Ungulates that Express HumanIgs

Approaches for the production of Homo H/L fetuses or calves aresummarized in FIG. 1. There are three schemes outlined therein. Thefirst relies on successive knockouts in regenerated fetal cell lines.This approach is the technically most difficult and has the highestlevel of risk but as noted above potentially yields faster results thanbreeding approaches. The other two schemes rely on breeding animals. Inthe second scheme, only single knockouts of heavy and light chain genesare required in male and female cell lines, respectively. This schemedoes not rely on regeneration of cell lines and is technically thesimplest approach but takes the longest for completion. Scheme 3 is anintermediate between schemes 1 and 2. In all schemes only Homo H/Lfetuses are generated because of potential difficulties in survival andmaintenance of Homo H/L knockout calves. If necessary, passiveimmunotherapy can be used to increase the survival of Homo H/L knockoutcalves.

Experimental Design The present invention preferably involves theproduction of a hemizygous male heavy chain knockout (M Hemi H) and ahemizygous female light chain knockout (F Hemi L) and the production of40 day fetuses from these targeted deletions. The cells from the embryosare harvested, and one allele of the light locus is targeted in the MHemi H cells and one allele of the heavy chain locus is targeted in theF Hemi L cells resulting in cells with hemizygous deletions of both theH and L loci (Hemi H/L). These cells are used to derive 40 day fetusesfrom which fibroblasts are isolated.

The M Hemi H/L fibroblasts are targeted with the other H chain allele tocreate M Homo H/Hemi L, and the F Hemi H/L are targeted with the other Lchain allele to create F Homo L/Hemi H. In order to create homozygousdeletions, higher drug concentrations are used to drive homozygoustargeting. However, it is possible that this approach may not besuccessful and that breeding may be necessary. An exemplary strategywhich relies on cre/lox targeting of the selection cassette allows thesame selective systems to be used for more than one targeted deletion.These fibroblasts are cloned and 40 day fetuses harvested and fibroblastcells isolated. The fetal cells from this cloning are targeted toproduce homozygous deletions of either the H or L loci resulting in MHomo H/L and F Homo H/L fetal fibroblasts. These fibroblasts are clonedand 40 day fetuses derived and fibroblasts isolated. The Homo H/L fetalfibroblasts are then used for incorporation of the HAC optionally by theuse of breeding procedures.

Library Construction Fetal fibroblast cells are used to construct agenomic library. Although it is reported to be significant that thetargeting construct be isogenic with the cells used for cloning, it isnot essential to the invention. For example, isogenic, substantiallyisogenic, or nonisogenic constructs may be used to produce a mutation inan endogenous immunoglobulin gene. In one possible method, Holsteincattle, which genetically contain a high level of inbreeding compared toother cattle breeds, are used. We have not detected any polymorphisms inimmunoglobulin genes among different animals. This suggests thatsequence homology should be high and that targeting with nonisogenicconstructs should be successful.

A library is constructed from one male cell line and one female cellline at the same time that the “clonability” testing is being conducted.It is envisioned that at the end of the process, a library will beproduced and a number of different fetal cell lines will be tested andone cell line chosen as the best for cloning purposes.

Genomic libraries are constructed using high molecular weight DNAisolated from the fetal fibroblast cells. DNA is size fractionated andhigh molecular weight DNA between 20-23 Kb is inserted into the lambdaphage vector LambdaZap or LambdaFix. The inventors have had excellentsuccess with Stratagene prepared libraries. Therefore, DNA is isolatedand the size selected DNA is sent to Stratagene for library preparation.To isolate clones containing bovine heavy and light chains, radiolabeledIgM cDNA and radiolabeled light chain cDNA is used. Additionally, lightchain genomic clones are isolated in case it is necessary to delete thelocus. Each fetal cell library is screened for bovine heavy and lightchain containing clones. It is anticipated that screening approximately10⁵-10⁶ plaques should lead to the isolation of clones containing eitherthe heavy chain or light chain locus. Once isolated, both loci aresubcloned into pBluescript and restriction mapped. A restriction map ofthese loci in Holsteins is provided in FIG. 2B (Knight et al. J Immunol140(10):3654-9, 1988). Additionally, a map from the clones obtained ismade and used to assemble the targeting construct.

Production of Targeting Constructs Once the heavy and light chain genesare isolated, constructs are made. The IgM construct is made by deletingthe IgM constant region membrane domain. As shown by Rajewsky andcolleagues in mice, deletion of the membrane domain of IgM results in ablock in B-cell development since surface IgM is a required signal forcontinued B-cell development (Kitamura et al., Nature 350:423-6). Thushomozygous IgM cattle lack B-cells. This should not pose a problem sincein the present strategy no live births of animals lacking functional Igare necessary. However, if necessary, passive immunotherapy may be usedto improve the survival of the animals until the last step when thehuman Ig loci are introduced.

An exemplary targeting construct used to effect knockout of the IgMheavy chain allele is shown below in FIG. 2A. For the heavy chain, themembrane IgM domain is replaced with a neomycin cassette flanked by loxP sites. The attached membrane domain is spliced together with the neocassette such that the membrane domain has a TAG stop codon insertedimmediately 5′ to the lox P site ensuring that the membrane domain isinactivated. This is placed at the 5′ end of the targeting constructwith approximately 5-6 kilobases of 3′ chromosomal DNA.

If increasing drug concentrations does not allow deletion of the secondallele of either IgM heavy or light chains, the cre/lox system (reviewedin Sauer, 1998, Methods 14:381-392) is used to delete the selectablemarker. As described below, the cre/lox system allows the targeteddeletion of the selectable marker. All selectable markers are flankedwith loxP sequences to facilitate deletion of these markers if thisshould be necessary.

The light chain construct contains the bovine lambda chain constantregion (e.g., the lambda light chain constant region found in Genbankaccession number AF396698 or any other ungulate lambda light chainconstant region) and a puromycin resistance gene cassette flanked by loxP sites and will replace the bovine gene with a puromycin cassetteflanked by lox P sites. Approximately 5-6 kilobases of DNA 3′ to thelambda constant region gene will be replaced 3′ to the puromycinresistance gene. The puromycin resistance gene will carry lox P sites atboth 5′ and 3′ ends to allow for deletion if necessary. Due to the highdegree of homology between ungulate antibody genes, the bovine lambdalight chain sequence in Genbank accession number AF396698 is expected tohybridize to the genomic lambda light chain sequence from a variety ofungulates and thus may be used in standard methods to isolate variousungulate lambda light chain genomic sequences. These genomic sequencesmay be used in standard methods, such as those described herein, togenerate knockout constructs to inactivate endogenous lambda lightchains in any ungulate.

A kappa light chain knockout construct may be constructed similarlyusing the bovine kappa light chain sequence in FIG. 3G or any otherungulate kappa light chain sequence. This bovine kappa light chain maybe used as a hybridization probe to isolate genomic kappa light chainsequences from a variety of ungulates. These genomic sequences may beused in standard methods, such as those described herein, to generateknockout constructs to inactivate endogenous kappa light chains in anyungulate.

Additional ungulate genes may be optionally mutated or inactivated. Forexample, the endogenous ungulate Ig J chain gene may be knocked out toprevent the potential antigenicity of the ungulate Ig J chain in theantibodies of the invention that are administered to humans. For theconstruction of the targeting vector, the cDNA sequence of the bovine IgJ chain region found in Genbank accession number U02301 may be used.This cDNA sequence may be used as a probe to isolate the genomicsequence of bovine Ig J chain from a BAC library such as RPC1-42 (BACPACin Oakland, Calif.) or to isolate the genomic sequence of the J chainfrom any other ungulate. Additionally, the human J chain coding sequencemay be introduced into the ungulates of present invention for thefunctional expression of human Igλ and IgM molecules. The cDNA sequenceof human J chain is available from Genbank accession numbers AH002836,M12759, and M12378. This sequence may be inserted into an ungulate fetalfibroblast using standard methods, such as those described herein. Forexample, the human J chain nucleic acid in a HAC, YAC vector, BACvector, cosmid vector, or knockin construct may be integrated into anendogenous ungulate chromosome or maintained independently of endogenousungulate chromosomes. The resulting transgenic ungulate cells may beused in the nuclear transfer methods described herein to generate thedesired ungulates that have a mutation that reduces or eliminates theexpression of functional ungulate J chain and that contain a xenogenousnucleic acid that expresses human J chain.

Additionally, the ungulate α-(1,3)-galactosyltransferase gene may bemutated to reduce or eliminate expression of thegalactosyl(α1,3)galactose epitope that is produced by theα-(1,3)-galactosyltransferase enzyme. If human antibodies produced bythe ungulates of the present invention are modified by this carbohydrateepitope, these glycosylated antibodies may be inactivated or eliminated,when administered as therapeutics to humans, by antibodies in therecipients that are reactive with the carbohydrate epitope. To eliminatethis possible immune response to the carbohydrate epitope, the sequenceof bovine alpha-(1,3)-galactosyltransferase gene may be used to design aknockout construct to inactive this gene in ungulates (Genbank accessionnumber J04989; Joziasse et al., J. Biol. Chem. 264(24):14290-7, 1989).This bovine sequence or the procine alpha-(1,3)-galactosyltransferasesequence disclosed in U.S. Pat. Nos. 6,153,428 and 5,821,117 may be usedto obtain the genomic alpha-(1,3)-galactosyltransferase sequence from avariety of ungulates to generate other ungulates with reduced oreliminated expression of the galactosyl(α1,3)galactose epitope.

If desired, the ungulate prion gene may be mutated or inactivated toreduce the potential risk of an infection such as bovine spongiformencephalopathy (BSE). For the construction of the targeting vector, thegenomic DNA sequence of the bovine prion gene may be used (Genbankaccession number AJ298878). Alternatively, this genomic prion sequencemay be used to isolate the genomic prion sequence from other ungulates.The prior gene may be inactivated using standard methods, such as thosedescribed herein or those suggested for knocking out thealpha-(1,3)-galactosyltransferase gene or prion gene in sheep (Denninget al., Nature Biothech., 19: 559-562, 2001).

For targeting the second allele of each locus, it may be necessary toassemble a new targeting construct containing a different selectablemarker, if the first selectable marker remains in the cell. As describedin Table 12, a variety of selection strategies are available and may becompared and the appropriate selection system chosen. Initially, thesecond allele is targeted by raising the drug concentration (forexample, by doubling the drug concentration). If that is not successful,a new targeting construct may be employed.

The additional mutations or the gene inactivation mentioned above may beincorporated into the ungulates of the present invention using variousmethodologies. Once a transgenic ungulate cell line is generated foreach desired mutation, crossbreeding may be used to incorporate theseadditional mutations into the ungulates of the present invention.Alternatively, fetal fibroblast cells which have these additionalmutations can be used as the starting material for the knockout ofendogenous Ig genes and/or the introduction of xenogenous Ig genes.Also, fetal fibroblast cells having a knockout mutation in endogenous Iggenes and/or containing xenogenous Ig genes can be uses as a startingmaterial for these additional mutations or inactivations.

Targeted Deletion of Ig Loci Targeting constructs are introduced intoembryonic fibroblasts, e.g., by electroporation. The cells whichincorporate the targeting vector are selected by the use of theappropriate antibiotic. Clones that are resistant to the drug of choicewill be selected for growth. These clones are then subjected to negativeselection with gancyclovir, which will select those clones which haveintegrated appropriately. Alternatively, clones that survive the drugselection are selected by PCR. It is estimated that it will be necessaryto screen at least 500-1000 clones to find an appropriately targetedclone. The inventors' estimation is based on Kitamura (Kitamura et al.,Nature 350:423-6, 1991) who found that when targeting the membranedomain of IgM heavy chain constant region approximately 1 in 300 neoresistant clones were properly targeted. Thus, it is proposed to poolclones into groups of 10 clones in a 96 well plate and screen pools of10 clones for the targeted clones of choice. Once a positive isidentified, single clones isolated from the pooled clone will bescreened. This strategy should enable identification of the targetedclone.

Because fibroblasts move in culture it is difficult to distinguishindividual clones when more than approximately ten clones are producedper dish. Further, strategies may be developed for clonal propagationwith high efficiency transfection. Several reasonable strategies, suchas dilution cloning, may be used.

Cre/Lox Excision of the Drug Resistance Marker As shown above, exemplarytargeting constructs contain selectable markers flanked by loxP sites tofacilitate the efficient deletion of the marker using the cre/loxsystem. Fetal fibroblasts carrying the targeting vector are transfectedvia electroporation with a Cre containing plasmid. A recently describedCre plasmid that contains a GFPcre fusion gene [Gagneten S. et al.,Nucleic Acids Res 25:3326-31 (1997)] may be used. This allows the rapidselection of all clones that contain Cre protein. These cells areselected either by FACS sorting or by manual harvesting of greenfluorescing cells via micromanipulation. Cells that are green areexpected to carry actively transcribed Cre recombinase and hence deletethe drug resistance marker. Cells selected for Cre expression are clonedand clones analyzed for the deletion of the drug resistance marker viaPCR analysis. Those cells that are determined to have undergone excisionare grown to small clones, split and one aliquot is tested in selectivemedium to ascertain with certainty that the drug resistance gene hasbeen deleted. The other aliquot is used for the next round of targeteddeletion. TABLE 12 Selectable markers and drugs for selection Gene DrugNeo^(r) G418¹ Hph Hygromycin B² Puro Puromycin³ Ecogpt Mycophenolicacid⁴ Bsr Blasticidin S⁵ HisD Histidinol⁶ DT-A Diphtheria toxin⁷¹Southern PJ, Berg P. 1982. Transformation of mammalian cells toantibiotic resistance with a bacterial gene under control of the SV40early region promoter. J Mol AppI Genet 1: 327-41.²Santerre RF, Allen NE, Hobbs JN Jr, Rao RN, Schmidt RJ. 1984.Expression of prokaryotic genes for hygromycin B and G418 resistance asdominant-selection markers in mouse L cells. Gene 30: 147-56.³Wirth M, Bode J, Zettlmeissl G, Hauser H. 1988. Isolation ofoverproducing recombinant mammalian cell lines by a fast and simpleselection procedure. Gene 73: 419-26.⁴Drews RE, Kolker MT, Sachar DS, Moran GP, Schnipper LE. 1996. Passageto nonselective media transiently alters growth of mycophenolicacid-resistant mammalian cells expressing the escherichia colixanthine-guanine phosphoribosyltransferase gene: implications forsequential selection strategies. Anal Biochem 235: 215-26.⁵Karreman C. 1998. New positive/negative selectable markers formammalian cells on the basis of Blasticidin deaminase-thymidine kinasefusions. Nucleic Acids Res 26: 2508-10.⁶Hartman SC, Mulligan RG. 1988. Two dominant-acting selectable markersfor gene transfer studies in mammalian cells. Proc Natl Acad Sci USA 85:8047-51.⁷Yagi T, Nada S., Watanabe N, Tamemoto H, Kohmura N, Ikawa Y, Aizawa S.1993. A novel negative selection for homologous recombinants usingdiphtheria toxin A fragment gene. Anal Biochem 214: 77-86.

Application of Targeting Strategies to Altering Immunoglobulin Genes ofOther Ungulates

To alter immunoglobulin genes of other ungulates, targeting vectors aredesigned to contain three main regions. The first region is homologousto the locus to be targeted. The second region is a drug selectionmarker that specifically replaces a portion of the targeted locus. Thethird region, like the first region, is homologous to the targeted locusbut is not contiguous with the first region in the wild type genome.Homologous recombination between the targeting vector and the desiredwild type locus results in deletion of locus sequences between the tworegions of homology represented in the targeting vector and replacementof that sequence with a drug resistance marker. In preferredembodiments, the total size of the two regions of homology isapproximately 6 kilobases, and the size of the second region thatreplaces a portion of the targeted locus is approximately 2 kilobases.This targeting strategy is broadly useful for a wide range of speciesfrom prokaryotic cells to human cells. The uniqueness of each vectorused is in the locus chosen for gene targeting procedures and thesequences employed in that strategy. This approach may be used in allungulates, including, without limitation, goats (Capra hircus), sheep(Ovis aries), and the pig (Sus scrufa), as well as cattle (Bos taurus).

The use of electroporation for targeting specific genes in the cells ofungulates may also be broadly used in ungulates. The general proceduredescribed herein is adaptable to the introduction of targeted mutationsinto the genomes of other ungulates. Modification of electroporationconditions (voltage and capacitance) may be employed to optimize thenumber of transfectants obtained from other ungulates.

In addition, the strategy used herein to target the heavy chain locus incattle (i.e., removal of all coding exons and intervening sequencesusing a vector containing regions homologous to the regions immediatelyflanking the removed exons) may also be used equally well in otherungulates. For example, extensive sequence analysis has been performedon the immunoglobulin heavy chain locus of sheep (Ovis aries), and thesheep locus is highly similar to the bovine locus in both structure andsequence (Genbank accession numbers Z71572, Z49180 through Z49188,M60441, M60440, AF172659 through AF172703). In addition to the largenumber of cDNA sequences reported for rearranged Ovis ariesimmunoglobulin chains, genomic sequence information has been reportedfor the heavy chain locus, including the heavy chain 5′ enhancer(Genbank accession number Z98207), the 3′ mu switch region (Z98680) andthe 5′ mu switch region (Z98681). The complete mRNA sequence for thesheep secreted form of the heavy chain has been deposited as accessionnumber X59994. This deposit contains the entire sequence of four codingexons, which are very homologous to the corresponding bovine sequence.

Information on the sheep locus was obtained from Genbank and used todetermine areas of high homology with bovine sequence for the design ofprimers used for PCR analysis. Because non-isogenic DNA was used totarget bovine cells, finding areas of high homology with sheep sequencewas used as an indicator that similar conservation of sequences betweenbreeds of cow was likely. Given the similarity between the sequences andstructures of the bovine and ovine immunoglobulin loci, it would beexpected that the targeting strategies used to remove bovineimmunoglobulin loci could be successfully applied to the ovine system.In addition, existing information on the pig (Sus scrofa, accessionnumber S42881) and the goat (Capra hircus, accession number AF140603),indicates that the immunoglobulin loci of both of these species are alsosufficiently similar to the bovine loci to utilize the present targetingstrategies.

EXAMPLE 15 Bovine IgM Knockout Removal of Exons 1-4 of Mu Heavy ChainLocus

The following procedures were used to generate bovine fibroblast celllines in which one allele of the immunoglobulin heavy chain (mu) locusis disrupted by homologous recombination. A DNA construct for effectingan IgM knockout was generated by the removal of exons 1-4 of the Mulocus (corresponds to IgM heavy chain gene) which were replaced with acopy of a neomycin resistance gene. Using this construct, neomycinresistant cell lines have been obtained which were successfully used innuclear transfer procedures, and blastocysts from these cell lines havebeen implanted into recipient cows. Additionally, some of theseblastocysts were tested to confirm that targeted insertion occurredappropriately in the mu locus using PCR procedures. Blastocystsresulting from nuclear transfer procedures from several of the celllines obtained indicated that heterozygous IgM-KO fetuses were ingestation. Additionally, both male and female cell lines that comprise asingle IgM heavy chain (mu) knockout have been produced. It isanticipated that mating of animals cloned from these cell lines willgive rise to progeny wherein both copies of mu are inactivated. Theseprocedures are discussed in greater detail below.

DNA Construct The DNA used in all transfections described in thisdocument was generated as follows. The four main exons (excluding thetransmembrane domain exons), CH1-4, are flanked by an XhoI restrictionsite at the downstream (CH4) end and an XbaI site at the upstream (CH1)end. The construct used for the transfection procedure consisted of 1.5kb of genomic sequence downstream of the XhoI site and 3.1 Kb of genomicsequence upstream of the XbaI site (FIGS. 3D and 3E). These sequenceswere isolated as described herein from a Holstein cow from a dairy herdin Massachusetts. A neomycin resistance marker was inserted betweenthese two fragments on a 3.5 Kb fragment, replacing 2.4 Kb of DNA,originally containing CH1-4, from the originating genomic sequence. Thebackbone of the vector was pBluescriptII SK+ (Stratagene) and the insertof 8.1 Kb was purified and used for transfection of bovine fetalfibroblasts. This construct is shown in FIGS. 3A-3C. Other mu knockoutconstructs containing other homologous regions and/or containing anotherantibiotic resistance gene may also be constructed using standardmethods and used to mutate an endogenous mu heavy chain gene.

Transfection/Knockout Procedures Transfection of fetal bovine wasperformed using a commercial reagent, Superfect Transfection Reagent(Qiagen, Valencia, Calif., USA), Catalog Number 301305.

Bovine fibroblasts were generated from disease-tested male Charlaiscattle at Hematech's Kansas facility and sent to Hematech's WorcesterMolecular Biology Labs for use in all experiments described. Any otherungulate breed, genus, or species may be used as the source of donorcells (e.g., somatic cells such as fetal fibroblasts). The donor cellsare genetically modified to contain a mutation that reduces oreliminates the expression of functional, endogenous Ig.

The medium used for culture of bovine fetal fibroblasts consisted of thefollowing components: 500 ml Alpha MEM (Bio-Whittaker #12-169F); 50 mlfetal calf serum (Hy-Clone #A-1111-D); 2 ml antibiotic/antimyotic(Gibco/BRL #15245-012); 1.4 ml 2-mercaptoethanol (Gibco/BRL #21985-023);5.0 ml L-Glutamine (Sigma Chemical #G-3126); and 0.5 ml tyrosinetartrate (Sigma Chemical #T-6134)

On the day prior to transfection procedures, cells were seeded in 60 mmtissue culture dishes with a targeted confluency of 40-80% as determinedby microscopic examination.

On the day of transfection, 5 μg of DNA, brought to a total volume of150 μl in serum-free, antibiotic-free medium, was mixed with 20 μl ofSuperfect transfection reagent and allowed to sit at room temperaturefor 5-10 minutes for DNA-Superfect complex formation. While the complexformation was taking place, medium was removed from the 60 mm tissueculture dish containing bovine fibroblasts to be transfected, and cellswere rinsed once with 4 ml of phosphate-buffered saline. One milliliterof growth medium was added to the 170 μl DNA/Superfect mixture andimmediately transferred to the cells in the 60 mm dish. Cells wereincubated at 38.5° C., 50% carbon dioxide for 2.5 hours. Afterincubation of cells with the DNA/Superfect complexes, medium wasaspirated off and cells were washed four times with 4 ml PBS. Five ml ofcomplete medium were added and cultures were incubated overnight at38.5° C., 5% CO₂. Cells were then washed once with PBS and incubatedwith one ml of 0.3% trypsin in PBS at 37° C. until cells were detachedfrom the plate, as determined by microscopic observation. Cells fromeach 60 mm dish were split into 24 wells of a 24 well tissue cultureplate (41.7 ul/well). One milliliter of tissue culture medium was addedto each well and plates were allowed to incubate for 24 hours at 38.5°C. and 5% CO₂ for 24 hours.

During all transfection procedures, sham transfections were performedusing a Superfect/PBS mixture containing no DNA, as none of those cellswould be expected to contain the neomycin resistance gene and all cellswould be expected to die after addition of G418 to the tissue culturemedium. This served as a negative control for positive selection ofcells that received DNA.

After the 24 hour incubation, one more milliliter of tissue culturemedium containing 400 μg G418 was added to each well, bringing the finalG418 concentration to 200 μg/ml. Cells were placed back into theincubator for 7 days of G418 selection. During that period, bothtransfected and sham transfection plates were monitored for cell deathand over 7 days, the vast majority of wells from the sham transfectionscontained few to no live cells while plates containing cells thatreceived the DNA showed excellent cell growth.

After the 7 day selection period, the cells from wells at 90-100%confluency were detached using 0.2 ml 0.3% trypsin in PBS and weretransferred to 35 mm tissue culture plates for expansion and incubateduntil they became at least 50% confluent, at which point, cells weretrypsinized with 0.6 ml 0.3% trypsin in PBS. From each 35 mm tissueculture plate, 0.3 ml of the 0.6 ml cell suspension was transferred to a12.5 cm² tissue culture flask for further expansion. The remaining 0.3ml was reseeded in 35 mm dishes and incubated until they attained aminimal confluency of approximately 50%, at which point cells from thoseplates were processed for extraction of DNA for PCR analysis. Flasksfrom each line were retained in the incubator until they had undergonethese analyses and were either terminated if they did not contain thedesired DNA integration or kept for future nuclear transfer andcryopreservation.

Screening for targeted integrations As described above the DNA sourcefor screening of transfectants containing the DNA construct was a 35 mmtissue culture dish containing a passage of cells to be analyzed. DNAwas prepared as follows and is adapted from a procedure published byLaird et al. (Laird et al., “Simplified mammalian DNA isolationprocedure”, Nucleic Acids Research, 19:4293). Briefly, DNA was preparedas follows. A cell lysis buffer was prepared with the followingcomponents: 100 mM Tris-HCl buffer, pH 8.5; 5 mM EDTA, pH 8.0; 0.2%sodium dodecyl sulfate; 200 mM NaCl; and 100 ug/ml Proteinase K.

Medium was aspirated from each 35 mm tissue culture dish and replacedwith 0.6 ml of the above buffer. Dishes were placed back into theincubator for three hours, during which time cell lysis and proteindigestion were allowed to occur. Following this incubation, the lysatewas transferred to a 1.5 ml microfuge tube and 0.6 ml of isopropanol wasadded to precipitate the DNA. Tubes were shaken thoroughly by inversionand allowed to sit at room temperature for 3 hours, after which the DNAprecipitates were spun down in a microcentrifuge at 13,000 rpm for tenminutes. The supernatant from each tube was discarded and the pelletswere rinsed with 70% ethanol once. The 70% ethanol was aspirated off andthe DNA pellets were allowed to air-dry. Once dry, each pellet wasresuspended in 30-50 ul of Tris (10 mM)-EDTA (1 mM) buffer, pH 7.4 andallowed to hydrate and solubilize overnight. 5-7 microliters of each DNAsolution was used for each polymerase chain reaction (PCR) procedure.

Two separate PCR procedures were used to analyze transfectants. Thefirst procedure used two primers that were expected to anneal to sitesthat are both located within the DNA used for transfection. The firstprimer sequence is homologous to the neomycin resistance cassette of theDNA construct and the second is located approximately 0.5 Kb away,resulting in a short PCR product of 0.5 Kb. In particular, primers Neo1(5′-CTT GAA GAC GAA AGG GCC TCG TGA TAC GCC-3′, SEQ ID NO: 42) andIN2521 (5′-CTG AGA CTT CCT TTC ACC CTC CAG GCA CCG-3′, SEQ ID NO: 43)were used. A Qiagen PCR kit was used for this PCR reaction. The PCRreaction mixture contained 1 pmole of each primer, 5 ul of 10× reactionbuffer, 10 ul of Q solution, 5 ul of DNA, and 1 ul of dNTP solution. Thereaction mixture was brought to a total volume of 50 ul with H₂O. ThisPCR amplification was performed using an initial denaturing incubationat 94° C. for two minutes. Then, 30 cycles of denaturation, annealing,and amplification were performed by incubation at 94° C. for 45 seconds,60° C. for 45 seconds, and 72° C. for two minutes. Then, the reactionmixture was incubated at 72° C. for five minutes and at 4° C. until themixture was removed from the PCR machine. Alternatively, any otherprimers that are homologous to the region of the knockout construct thatintegrates into the genome of the cells may be used in a standard PCRreaction under appropriate reaction conditions to verify that cellssurviving G418 selection were resistant as a result of integration ofthe DNA construct.

Because only a small percentage of transfectants would be expected tocontain a DNA integration in the desired location (the Mu locus),another pair of primers was used to determine not only that the DNAintroduced was present in the genome of the transfectants but also thatit was integrated in the desired location. The PCR procedure used todetect appropriate integration was performed using one primer locatedwithin the neomycin resistance cassette of the DNA construct and oneprimer that would be expected to anneal over 1.8 Kb away, but only ifthe DNA had integrated at the appropriate site of the IgM locus (sincethe homologous region was outside the region included in the DNAconstruct used for transfection). The primer was designed to anneal tothe DNA sequence immediately adjacent to those sequences represented inthe DNA construct if it were to integrate in the desired location (DNAsequence of the locus, both within the region present in the DNAconstruct and adjacent to them in the genome was previously determined).In particular, primers Neo1 and OUT3570 (5′-CGA TGA ATG CCC CAT TTC ACCCAA GTC TGT C-3′, SEQ ID NO: 44) were used for this analysis. This PCRreaction was performed using a Qiagen PCR kit as described above for thefirst PCR reaction to confirm the integration of the targeting constructinto the cells. Alternatively, this PCR analysis may be performed usingany appropriate reaction conditions with any other primer that ishomologous to a region of the knockout construct that integrates intothe genome of the cells and any other primer that is homologous to aregion in the genome of the cells that is upstream or downstream of thesite of integration.

Using these methods, 135 independent 35 mm plates were screened fortargeted integration of the DNA construct into the appropriate locus. Ofthose, DNA from eight plates was determined to contain an appropriatelytargeted DNA construct and of those, three were selected for use innuclear transfer procedures. Those cells lines were designated as“8-1C”, “5-3C” and “10-1C”. Leftover blastocysts not used for transferinto recipient cows were used to extract DNA which was subjected toadditional PCR analysis. This analysis was effective using a nested PCRprocedure using primers that were also used for initial screening oftransfected lines.

As noted above, three cell lines were generated using the gene targetingconstruct designed to remove exons 1-4 of the mu locus. These lines alltested positive for targeted insertions using a PCR based test and wereused for nuclear transfers. Leftover blastocysts resulting from thosenuclear transfers were screened by PCR testing the appropriatelytargeted construct. The following frequencies of positive blastocystswere obtained:

Cell Line 8-1C: 6/8

Cell Line 10-1C: 2/16

Cell Line 5-3C: 0/16

Although at forty days of gestation, 11 total pregnancies were detectedby ultrasound, by day 60, 7 fetuses had died. The remaining 4 fetuseswere processed to regenerate new fetal fibroblasts and remaining organswere used to produce small tissue samples for PCR analysis. The resultsof the analyses are below:

Line 8-1C: two fetuses, one fetus positive for targeted insertion by PCR

Line 10-1C: one fetus, positive for targeted insertion by PCR

Line 5-3C: one fetus, negative for targeted insertion by PCR

Surprisingly, although the frequency of 10-1C blastocysts testingpositive for targeted insertion was only 2/16, the one viable 60-dayfetus obtained from that cell line was positive as determined by PCR. Apositive fetus from 8-1C was also obtained. Southern blot analysis ofDNA of all tissue samples is being effected to verify that the constructnot only targeted correctly at one end (which is determined by PCR ofthe shorter region of homology present in the original construct) butalso at the other end. Based on results to date, it is believed that twoheavy chain knockout fetuses from two independent integration eventshave been produced. Also, since these fetuses were derived from twodifferent lines, at least one is likely to have integrated constructcorrectly at both ends. Once the Southern blot analyses have confirmedappropriate targeting of both ends of targeting construct, furthernuclear transfers will be performed to generate additional fetuses whichwill be carried to term.

Nuclear Transfer and Embryo Transfer Nuclear transfers were performedwith the K/O cell line (8-1-C (18)) and eight embryos were produced. Atotal of six embryos from this batch were transferred to three diseasefree recipients at Trans Ova Genetics (“TOG”; Iowa).

Frozen embryos have been transferred to ten disease free recipients toobtain disease free female fibroblast cell lines. Fetal recoveries arescheduled after confirming the pregnancies at 35-40 days.

Pregnancy Diagnosis and Fetal Recovery Pregnancy status of the eighteenrecipients transferred with cloned embryos from knockout fetal cells waschecked by ultrasonography. The results are summarized below. TABLE 13Pregnancy at 40 days using mu heavy chain knockout donor cells Clone IDNo of recips transferred Pregnancy at 40 days (%) 8-1-0C 5 4 (80) 10-1-C6 4 (67) 5-3-C 5 3 (60) Total 16 11 (69) 

Pregnancy Diagnosis Pregnancy status of the three recipients to whomcloned embryos were transferred from knockout cells (8-1C) was checked;one was open and the other two required reconfirmation after one month.

Fetal Recoveries and Establishment of Cell Lines Eleven pregnancies withthe K/O embryos at 40 days were obtained. Four live fetuses were removedout of these at 60 days. Cell lines were established from all four andcryopreserved for future use. Also we collected and snap froze tissuesamples from the fetuses and sent them to Hematech molecular biologylaboratory for PCR/Southern blot analysis.

All four of the cell lines were male. In order to secure a female cellline, cell lines were established and cryopreserved for futureestablishment of K/O cells from the fetuses (six) collected at 55 daysof gestation from the pregnancies established at Trans Ova Genetics withdisease free recipients. Recently, the existence of a female cell linecontaining a mu knockout was confirmed. This female cell line may beused to produce cloned animals which may be mated with animals generatedfrom the male cell lines, and progeny screened for those that containthe double mu knockout.

If desired, a cell from the resulting knockout fetus or knockoutoffspring can be used in a second round of nuclear transfer to generateadditional cloned offspring. Cells from the initial knockout fetus orknockout offspring may also be frozen to form a cell line to be used asa source of donor cells for the generation of additional knockoutungulates.

Insertion of Transcription Termination Sequence into Mu Heavy ChainLocus

Bovine fibroblast cell lines in which one allele of Igμ locus is mutatedby insertion of a transcription termination sequence were generated byhomologous recombination. In particular, transcription of functional,full-length Igμ mRNA was prevented by inserting a neomycin orpuromycin-resistance gene (neo or puro, described herein) and atranscription termination cassette (STOP) in exon 2. Thus, the resultingimmature Igμ transcripts lack the functional domain. For this method, aDNA targeting construct containing a puro gene (i.e., the CμKOpurovector) was electroporated into bovine fibroblast cell lines, and thenpuromycin-resistant colonies were isolated. Based on PCR analysis,homologous recombination in exon 2 occurred in some colonies. Thus,bovine fibroblast cell lines in which one allele of the Igμ locus ismutated were generated. From the hemizygously mutated (hemi-IgμKO)fibroblasts, three fetuses in which one allele of the Igμ locus ismutated were generated and the hemi-IgμKO fibroblasts werereestablished. Then, a DNA targeting construct containing a neo gene(i.e., the CμKOneo vector) was electroporated into the hemi-IgμKOfibroblasts, and neomycin-resistant colonies were isolated. Based on PCRanalysis, homologous recombination in exon 2 of the remaining alleleoccurred in some colonies. Thus, bovine fibroblast cell lines in whichboth alleles of the Igμ locus are mutated were generated. From thehomozygously mutated (homo-IgμKO) fibroblasts, five fetuses in whichboth alleles of Igμ locus are mutated were generated, and the homo-IgμKOfibroblasts were reestablished. In this way, bovine fibroblast celllines in which both alleles of Igμlocus are mutated (homo-IgμKO) weregenerated. Alternatively, homo-IgμKO fibroblasts can be generated usingthe same knockout vector that was used to produce hemizygous knockoutcells and a higher concentration of antibiotic to select for homozygousknockout cells. Homo-IgμKO calves can be generated from the homo-IgμKOfibroblast cell lines, using either standard nuclear transfer methods orany of the nuclear or chromatin transfer methods described herein.

These methods are described further below.

Construction of Igμ KO vectors The Igμ KO vectors were generated asfollows (FIGS. 40A-40D). To isolate genomic DNA around exon 2 of the Igμgene, a DNA probe was amplified by PCR using the following primer pair5′-TGGTCACTCCAAGTGAGTCG-3′ (SEQ ID NO: 69) and5′-TGGAGTGAAATCAGGTGAAGG-3′ (SEQ ID NO: 70). Using this probe, a bovine(Holstein) genomic λ phage library was screened, and four positive λphage clones were identified. One clone out of the four clones wasanalyzed further by restriction mapping. The 9 kilobases of XhoI-BamHIgenomic fragment containing all of the Cμ exons was subcloned intopBluescript II SK(−) in which the KpnI site is already replaced withSrfI site. Then, both the puro and STOP cassettes were inserted at theBglII site, which is just located in exon 2 of Cμ. The orientation ofboth puro and STOP cassettes was a sense strand-orientation relative tothe Igμ gene. A diphtheria toxin gene (DT-A, Gibco) was then added tothe Not I site in the pBluescript II SK(−). DT-A was inserted in forwardorientation relative to the puromycin-resistance gene in the targetingcassette to kill cells in which the targeting cassette was randomlyintegrated in the genome (pBCμΔKOpuro vector). Similarly, another KOvector containing neo gene was constructed (pBCμΔNKOneo vector). In someembodiments, the vector contains a stretch of DNA adjacent to the DT-Anegative selection marker to protect the negative selection marker froma possible nuclease attack.

Transfection/Knockout Procedures Transfection of fetal fibroblast celllines (Holstein) was performed using the following standardelectroporation protocol. The medium used to culture the bovine fetalfibroblasts contained 500 ml Alpha MEM (Gibco, 12561-049), 50 ml fetalcalf serum (Hy-Clone #ABL13080), 5 ml penicillin-streptomycin (SIGMA),and 1 ml 2-mercaptoethanol (Gibco/BRL #21985-023). On the day prior totransfection, cells were seeded on a T175 tissue culture flask with aconfluency of 80-100%, as determined by microscopic examination. On theday of transfection, about 10⁷ bovine fibroblasts cells were trypsinizedand washed once with alpha-MEM medium. After resuspension of the cellsin 800 μl of alpha-MEM, 30 μg of the Srf I-digested KO vector(pB.CμΔKOpuro vector) dissolved in Hepes buffer saline (HBS) containing1 mM spermidine was added to the cell suspension and mixed well bypipetting. The cell-DNA suspension was transferred into anelectroporation cuvette and electroporated at 550 V and 50 μF. Afterthat, the electroporated cells were plated onto thirty 48-well plateswith the alpha-MEM medium supplemented with the serum. After a 48hour-culture, the medium was replaced with medium containing 1 μg/ml ofpuromycin, and the cells were cultured for 2-3 weeks to select puromycinresistant cells. After selection, all colonies which reached close to100% confluency were divided into two replica plates (24-well and48-well plates): one for genomic DNA extraction, and the other plate fornuclear transfer. Genomic DNA was extracted from the colonies to screenfor the desired homologous recombination events by PCR.

Screening for targeted integrations As described above, the genomic DNAwas independently extracted from each 24-well using the PUREGENE DNAisolation Kit (Gentra SYSTEMS) according to the manufacture's protocol.Each genomic DNA sample was resuspended in 20 μl of 10 mM Tris-Cl(pH8.0) and 1 mM EDTA (EDTA). Screening by PCR was performed using thefollowing primer pair “F14” (5′-ccacaaaggaaaaagctgcactgctatac-3′; SEQ IDNO: 71) and “R14” (5′-tgtgggatcaggaggtcagatagacatc-3′; SEQ ID NO: 72).The sequence of one primer is located in the KO vector, and the sequenceof the other primer is located just outside of the integrated vector inthe targeted endogenous locus (FIGS. 40A and 40B). Therefore, theexpected PCR product is detected only when the KO vector is integratedinto the targeted locus by homologous recombination. The PCR reactionmixtures contained 17.9 μl water, 3 μl of 10×LA PCR buffer II (Mg²⁺plus), 4.8 μl of dNTP mixture, 10 pmol of forward primer, 10 pmol ofreverse primer, 2 μl of genomic DNA, and 0.3 μl of LA Taq. Forty cyclesof PCR were performed by incubating the reaction mixtures under thefollowing conditions: 85° C. for three minutes, 94° C. for one minute,98° C. for 10 seconds, and 68° C. for five minutes. After PCR, thereaction mixtures were analyzed by electrophoresis. Out of 423 screenedclones, two clones (#147 and #384) generated the expected PCR products.The identity of these PCR products was confirmed by sequencing. Based onthe presence of a polymorphic marker, the KO vector was integrated into“allele A” and “allele B” of Cμ exon 2 in clones #384 and #147,respectively. These two clones were used as donor cells to generatefetuses as described below.

Chromatin transfer In vitro-matured oocytes were enucleated at 20 hpm.Bovine Ig mu knockout clones were trypsinized and washed in Ca/Mg Hank'sBalanced Salt Solution (HBSS) and permeabilized by incubation of50,000-100,000 cells in 31.25 units Streptolysin O (SLO-Sigma, St.Louis, Mo.) in 100 μl for 30 minutes in a 37° C. H₂O bath. Cell sampleswere incubated with propidium iodide and observed by florescentmicroscopy to monitor permeabilization based on uptake of the dye.

Permeabilized fibroblasts were washed, pelleted, and incubated in 40 μlof mitotic extract prepared from MDBK cells containing an ATP-generatingsystem (1 mM ATP, 10 mM creatine phosphate, and 25 μg/ml creatinekinase) for 30 minutes in a 37° C. H₂O bath. Cell samples were stainedwith Hoechst 33342 and observed by florescent microscopy to monitorchromatin condensation. At the end of incubation, the reaction mix wasdiluted with 500 μl cell culture media (Alpha MEM with 10% FBS). Thesecells were pelleted and resuspended in TL Hepes and used for chromatintransfer in enucleated oocytes as described herein. Three fetuses(#2184-1, 2184-2, 3287) were determined to be hemizygous Igμ KO fetusesin which the puroKO vector is integrated into one allele of the Igμgene. Fetuses #2184-1 and 2184-2 were derived from clone #384, and fetus#3287 was derived from clone #147. Thus, three bovine fibroblast celllines in which one allele of the Igμ locus is mutated by the KO vectorwere successfully generated. TABLE 14 Pregnancies, fetal recovery, andcell lines with hemizygous Igμ KO clones derived from primary Holsteinfibroblast cell line 6939 No of Igμ Clone Total Blastocyst No ofPregnant at 40 d Pregnant at 60 d No of fetuses hemizygous ID CTs (%)recipients (%) (%) recovered KO fetuses 147 188 20 (15) 14 7 (50) 2 (14)2 (14) 1 (#3287) 384 234 35 (22) 16 8 (50) 4 (25) 7* (25)  2 (#2184-1,#2184-2) Total 422 55 (19) 30 15 (50)  6 (20) 9 (30) 3*Three sets of twins. Three correctly targeted hemizygous Igμ KO fetuseswere identified (2184-1, 2184-2 and 3287).

2^(nd) Transfection/Knockout Procedures Transfection of the hemi-IgμKOfetal fibroblast cell lines was performed using a similar method. Clone#3287 in which the puroKO vector is integrated into the “allele B” wasextensively used for obtaining homo-IgμKO clones. The medium used toculture the bovine fetal fibroblasts contained 500 ml Alpha MEM (Gibco,12561-049), 50 ml fetal calf serum (Hy-Clone #ABL13080), 5 mlpenicillin-streptomycin (SIGMA), and 1 ml 2-mercaptoethanol (Gibco/BRL#21985-023). On the day prior to transfection, cells were seeded on aT175 tissue culture flask with a confluency of 80-100%, as determined bymicroscopic examination. On the day of transfection, about 10⁷ bovinefibroblasts cells were trypsinized and washed once with alpha-MEMmedium. After resuspension of the cells in 800 μl of alpha-MEM, 30 μg ofthe Srf I-digested KO vector (pBCμΔNKOneo vector) dissolved in HBScontaining 1 mM spermidine was added to the cell suspension and mixedwell by pipetting. The cell-DNA suspension was transferred into anelectroporation cuvette and electroporated at 550 V and 50° F. Afterthat, the electroporated cells were plated onto thirty 48-well plateswith the alpha-MEM medium supplemented with the serum. After a 48 hourculture, the medium was replaced with medium containing 500 μg/ml ofG418, and the cells were cultured for 2-3 weeks to select G418 resistantcells. After selection, all colonies that reached close to 100%confluency were divided into two replica plates (24-well and 48-wellplates): one plate for genomic DNA extraction, and the other plate fornuclear transfer. Genomic DNA was extracted from the colonies to screenfor the desired homologous recombination events by PCR.

Screening for homozygously targeted integrations As described above, thegenomic DNA was independently extracted from each 24-well independentlyusing the PUREGENE DNA isolation Kit (Gentra SYSTEMS) according to themanufacture's protocol. Each genomic DNA sample was resuspended in 20 μlof 10 mM Tris-Cl (pH8.0) and 1 mM EDTA (EDTA). Screening by PCR wasperformed using the following primer pair “neoF3”(5′-TTTGGTCCTGTAGTTTGCTAACACACCC-3′; SEQ ID NO: 73) and “neoR3”(5′-GGATCAGTGCCTATCACTCCAGGTTG-3′; SEQ ID NO: 74). The sequence of oneprimer is located in the KO vector, and the sequence of the other primeris located just outside of the integrated vector in the targetedendogenous locus (FIGS. 40C and 40D). Therefore, the expected PCRproduct is detected only when the KO vector is integrated into thetargeted locus by homologous recombination. The PCR reaction mixturescontained 17.9 μl water, 3 μl of 10×LA PCR buffer II (Mg²⁺ plus), 4.8 μlof dNTP mixture, 10 pmol of forward primer, 10 pmol of reverse primer, 2μl of genomic DNA, and 0.3 μl of LA Taq. Forty cycles of PCR wereperformed by incubating the reaction mixtures under the followingconditions: 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, and 68° C. for seven minutes. After PCR, the reactionmixtures were analyzed by electrophoresis. Out of 569 screened clones,seven clones (#76, 91, 184, 442, 458, 496, and 527) produced theexpected PCR products, which were confirmed by sequencing. Based on thepresence of a polymorphic marker, the KO vector integrated into “alleleA” of Cμ exon 2 in all the clones except #184. Thus, the puroKO vectorand the neoKO vector integrated into “allele B” and “allele A,”respectively, of six homozygous Igμ KO clones. Four clones (#76, 91,442, 458) were used as donor cells to generate fetuses as describedbelow.

Chromatin transfer In vitro-matured oocytes were enucleated at 20 hpm.Bovine Ig mu knockout clones were trypsinized and washed in Ca/Mg Hank'sBalanced Salt Solution (HBSS) and permeabilized by incubation of50,000-100,000 cells in 31.25 units Streptolysin O (SLO-Sigma, St.Louis, Mo.) in 100 μl for 30 minutes in a 37° C. H₂O bath. Cell sampleswere incubated with propidium iodide and observed by florescentmicroscopy to monitor permeabilization based on uptake of the dye.

Permeabilized fibroblasts were washed, pelleted, and incubated in 40 μlof mitotic extract prepared from MDBK cells containing an ATP-generatingsystem (1 mM ATP, 10 mM creatine phosphate, and 25 μg/ml creatinekinase) for 30 minutes in a 37° C. H₂O bath. Cell samples were stainedwith Hoechst 33342 and observed by florescent microscopy to monitorchromatin condensation. At the end of incubation, the reaction mix wasdiluted with 500 μl cell culture media (Alpha MEM with 10% FBS). Thesecells were pelleted and resuspended in TL Hepes and used for chromatintransfer in enucleated oocytes as described herein. Five fetuses (#4658,3655, 5109, 5139, and 4554) from clone #75 and three fetuses (#4039-2,5133-2, and 5112) from clone #91 are homozygous Igμ KO fetuses in whichthe puroKO vector and the neoKO vector are integrated into “allele B”and “allele A,” respectively. Thus, eight bovine fibroblast cell linesin which both alleles of the Igμ locus are mutated by the KO vectorswere successfully generated. TABLE 15 Embryo development and transferswith homozygous Igμ clones from hemizygous line 3287 No of No of No ofIgM Total Blastocysts recipients Pregnant at 40 d fetuses homozygousClone ID CTs (%) implanted (%) recovered KO fetuses  91 1019 254 (36) 53 18 (34)  14* 3 442 249 50 (29) 06 5 (83)  7* 0 496 240 70 (42) 0 0(0)  0 0  76 141 20 (20) 09 6 (67) 5 5 458 32  2 (09) 01  1 (100) 1 0Total 1681 396 (34) 69 30 (43)  27  8Two sets of twins were produced with each clone. Eight correctlytargeted homozygous Igμ KO fetuses identified (#4658, 3655, 5109, 5139,4554, 4039-2, 5133-2, and 5112).

EXAMPLE 16 Optional Immunodepletion of Endogenous Antibodies Productionof Antibodies Reactive with Endogenous Ungulate Antibodies

For the preparation of polyclonal antibodies reactive with endogenousungulate antibodies or B-cells, one or more ungulate antibodies (e.g.,polyclonal ungulate immunoglobulin, IgG, or IgM), fragments of ungulateantibody proteins (e.g., mu heavy chain, kappa light chain, or lambdalight chain), or fusion proteins containing defined portions of ungulateantibodies can be purified from natural sources (e.g., serum samples orcultures of ungulate B-cells) or synthesized in, e.g., mammalian,insect, or bacterial cells by expression of corresponding DNA sequencescontained in a suitable cloning vehicle. Fusion proteins are commonlyused as a source of antigen for producing antibodies. Alternatively,mixtures of ungulate antibodies, such as polyclonal ungulateimmunoglobulin, can be used as the antigen source. The ungulateantibodies can be optionally purified, and then coupled to a carrierprotein, mixed with Freund's adjuvant to enhance stimulation of theantigenic response in an inoculated animal, and injected into otherungulates, rabbits, mice, or other laboratory animals. Primaryimmunizations are carried out with Freund's complete adjuvant andsubsequent immunizations performed with Freund's incomplete adjuvant.Following booster injections at bi-weekly intervals, the inoculatedanimals are then bled and the sera isolated. The sera is used directlyor is purified prior to use by various methods, including affinitychromatography employing reagents such as Protein A-Sepharose,antigen-Sepharose, and anti-horse-1 g-Sepharose. Antibody titers can bemonitored by Western blot and immunoprecipitation analyses usingungulate antibodies. Immune sera can be affinity purified using ungulateantibodies coupled to beads. Antiserum specificity can be determinedusing a panel of xenogenous antibodies (e.g., human antibodies),ungulate IgG, and ungulate IgM molecules.

Alternatively, monoclonal antibodies are produced by removing the spleenfrom the inoculated animal, homogenizing the spleen tissue, andsuspending the spleen cells suspended in phosphate buffered saline(PBS). The spleen cells serve as a source of lymphocytes, some of whichproduce antibody of the appropriate specificity. These cells are thenfused with permanently growing myeloma partner cells, and the productsof the fusion plated into a number of tissue culture wells in thepresence of selective agents, such as hypoxanthine, aminopterine, andthymidine (Mocikat, J. Immunol. Methods 225:185-189, 1999; Jonak et al.,Hum. Antibodies Hybridomas 3:177-185, 1992; Srikumaran et al., Science220:522, 1983. The wells can then be screened by ELISA to identify thosecontaining cells making antibody capable of binding to ungulateantibodies, fragments, or mutants thereof. These cells can then bere-plated and, after a period of growth, the wells containing thesecells can be screened again to identify antibody-producing cells.Several cloning procedures can be carried out until over 90% of thewells contain single clones that are positive for specific antibodyproduction. From this procedure, a stable line of clones that producethe antibody can be established. The monoclonal antibody can then bepurified by affinity chromatography using Protein A Sepharose andion-exchange chromatography, as well as variations and combinations ofthese techniques. Once produced, monoclonal antibodies are also testedfor specific ungulate antibody recognition by ELISA, Western blot,and/or immunoprecipitation analysis (see, e.g., Kohler et al., Nature256:495, 1975; Kohler et al., European Journal of Immunology 6:511,1976; Kohler et al., European Journal of Immunology 6:292, 1976;Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas,Elsevier, New York, N.Y., 1981; Ausubel et al., supra).

As an alternate or adjunct immunogen to an ungulate antibody, peptidescorresponding to relatively unique hydrophilic regions of an ungulateantibody can be generated and coupled to keyhole limpet hemocyanin (KLH)through an introduced C-terminal lysine. Antiserum to each of thesepeptides can be similarly affinity-purified on peptides conjugated toBSA, and specificity tested by ELISA and Western blotting using peptideconjugates, and by Western blotting and immunoprecipitation usingxenogenous and ungulate antibodies.

Antibodies of the invention can be produced using ungulate antibodyamino acid sequences that do not reside within highly conserved regions,and that appear likely to be antigenic, as evaluated by criteria such asthose provided by the Peptide Structure Program (Genetics Computer GroupSequence Analysis Package, Program Manual for the GCG Package, Version7, 1991) using the algorithm of Jameson et al., CABIOS 4:181, 1988.These fragments can be generated by standard techniques, e.g., by thePCR, and cloned into any appropriate expression vector. For example, GSTfusion proteins can be expressed in E. coli and purified using aglutathione-agarose affinity matrix (Ausubel et al., supra). To generatehorse polyclonal antibodies, and to minimize the potential for obtainingantisera that is non-specific, or exhibits low-affinity binding to anungulate antibody, two or three fusions may be generated for eachfragment injected into a separate animal. Antisera are raised byinjections in series, preferably including at least three boosterinjections.

In addition to intact monoclonal and polyclonal anti-ungulateantibodies, various genetically engineered antibodies and antibodyfragments (e.g., F(ab′)2, Fab′, Fab, Fv, and sFv fragments) can beproduced using standard methods. Truncated versions of monoclonalantibodies, for example, can be produced by recombinant methods in whichplasmids are generated that express the desired monoclonal antibodyfragment(s) in a suitable host.

Ladner (U.S. Pat. Nos. 4,946,778 and 4,704,692) describes methods forpreparing single polypeptide chain antibodies. Ward et al., Nature341:544-546, 1989, describes the preparation of heavy chain variabledomain which have high antigen-binding affinities. McCafferty et al.,Nature 348:552-554, 1990, show that complete antibody V domains can bedisplayed on the surface of fd bacteriophage, that the phage bindspecifically to antigen, and that rare phage (one in a million) can beisolated after affinity chromatography. Boss et al., U.S. Pat. No.4,816,397, describes various methods for producing immunoglobulins, andimmunologically functional fragments thereof, that include at least thevariable domains of the heavy and light chains in a single host cell.Cabilly et al., U.S. Pat. No. 4,816,567, describes methods for preparingchimeric antibodies. In addition, the antibodies can be coupled tocompounds, such as toxins or radiolabels. An exemplary anti-bovine lightchain antibody has been previously described (Goldsby et al., Vet.Immunol. Immunopath. 17: 25, 1987).

Exemplary Methods for Producing Equine Antibodies that are Reactive withBovine Antibodies

Antibodies against bovine immunoglobulin can be made in many species,including the equine, caprine, ovine, porcine, or bovine. A purifiedsample of bovine antibody is emulsified in an adjuvant solution such asalum or Freund's adjuvant in a ratio of 3 parts adjuvant solution to 1part antibody. The solution is injected subcutaneously in each shoulderand in each side of the neck of the horse. Each site of injectionreceives 0.25 mg of antibody. A second boost of emulsified antibodyadjuvant solution is similarly given at one month after the firstimmunization. Additional boosts can be given to maintain high antibodytiters.

The horse is bled starting at two weeks after the first boost. Bleedingis performed using standard methods which consist of restraining thehorse in an appropriate shoot and inserting a needle into the jugularvein which runs beneath the skin on either side of the esophagus. Thetotal blood volume is approximately 6% of body weight and 15% of theblood volume can be collected every two weeks.

Blood is allowed to clot, and the sample is spun in a centrifuge toseparate the clot. The liquid serum fraction is decanted. Alternatively,clotting is prevented with either heparin or EDTA, and the cellsfraction is pelleted by centrifugation and the plasma is decanted.Several standard methods can be used to prepare a partially purifiedsample of antibody from either plasma or serum. Exemplary methodsinclude the standard Kohn fractionation system, affinity chromatographywith Staphylococcus aureous protein A, and ion exchange chromatography.

Equine antibody may be preabsorbed against human immunoglobulin toensure that equine antibodies which cross react with human antibody areremoved. This step can be performed using standard procedures such aspassing the equine antibody fraction through an affinity column made byattaching human immunoglobulin to a solid support. This step ensuresthat administration of the equine antibody to a bovine expressing humanantibody does not eliminate the desired B-cells expressing humanantibody.

Alternatively, if human expressing B-cells are eliminated by the equineantibody, B-cells or B-cell precursors from another animal (e.g., ahuman) can be administered to the bovine during its fetal stage or afterit is born (see, for example, WO 01/35735, filed Nov. 17, 2000).

Exemplary Methods for Immunodepleting Endogenous Ungulate Antibodies

B-cell immunodepletion may be performed by injecting the ungulate (e.g.,a bovine, such as a newborn calf), with equine immunoglobulin againstbovine antibody. Immediately after birth the calf is eitherintravenously infused with between 1 mg and 1 gram (e.g., between 10 and100 mg) of antibody or the antibody is given orally. Antibody ispreferably given up to 12 hours prior to nursing or administration ofcolostrum. Additionally or alternatively, the antibody is administeredafter nursing or administration of colostrum.

The success of immunodepletion of bovine antibody expressing B-cells canbe monitored by several methods. Blood samples can be collected andB-cells can be analyzed by FACS to determine the proportion of B-cellsproducing bovine antibodies, human antibodies, or chimeric bovine/humanantibodies. In this assay, fluorescently labeled anti-bovine Igantibodies are used to bind Ig molecules expressed on the surface of theB-cells, and the number of B-cells labeled with these antibodies isdetermined using FACS. The amount of antibodies secreted by B-cells isdetermined using a standard ELISA capture assay with an anti-bovine Igantibody. Similar methods can be used to measure human antibody levels.Blood, milk, or lymph samples may be taken at various time points, suchas 1, 2, or 3 times a week, to measure the residual endogenous B-cellactivity. Preferably, the amount of endogenous antibodies secreted byB-cells, is reduced by at least 25, 50, 75, or 90% compared to thecorresponding amount in the absence of treatment with an antibodyreactive with endogenous B-cells or endogenous antibody. Achieving thislevel of inhibition may require a few days, a few weeks, or longerdepending upon the dose and dosing frequency of the particular compoundthat is administered to the calves. If necessary, larger doses or morefrequent dosing schemes than those mentioned above may also be used tofurther reduce the level of endogenous B-cell activity or to cause thedesired reduction in activity to be achieved sooner. As the bovineantibodies derived from colostrums are depleted, the level of humanantibody should increase.

Additional immunodepletion can be performed to further reduce thepopulation B-cells expressing of bovine antibody. As the animal grows,substantially higher amounts of antibody are required to ensuresuccessful B-cell depletion.

For the isolation of the xenogenous antibodies, blood, milk, or lymphsamples are taken from the calves at multiple intervals, such as everyday for 1, 3, 5, 7, 14, or more days, and used in standard methods forthe purification of the xenogenous antibody. If desired, blood samplesmay also be analyzed for continued inhibition of the production ofendogenous antibodies by the calves.

Inhibition of B-Cell Development in Fetuses

An anti-bovine IgM antibody (das6) was injected into fetuses todemonstrate the ability of an antibody to inhibit the development ofB-cells in fetuses. Similar results are expected for fetuses thatcontain a nucleic acid encoding a xenogenous antibody. Three fetuses atday 75 of gestation were injected with 1.5 mg of the anti-bovine IgMantibody. For this injection, the standard surgical procedure describedabove was used to inject the antibody into the peritoneal cavity of thefetuses. A similar injection procedure was performed on two controlfetuses, which were injected with either 3×10⁷ fetal liver cells (FIGS.28A and 28F) or 1.8×10⁷ mouse bone marrow cells (FIGS. 28B and 28G),which should not affect the number of B-cells in the fetuses. Afterapproximately 41 days, the three experimental and two control fetuseswere removed from the pregnant cows. Standard methods were used toisolate peripheral blood lymphocytes from blood samples from each of thefetuses.

To determine the percentage of peripheral blood lymphocytes that wereB-cells, these lymphocytes were analyzed using standard FACS analysisfor the expression of either IgM or antibody light chain molecules,which are both expressed on the surface of B-cells. As illustrated inFIG. 28A and FIG. 28B, approximately 19.82 to 26.61% of the peripheralblood lymphocytes from the control fetuses expressed IgM. In contrast,only 7.78, 11.80, or 3.95% of the peripheral blood lymphocytes from thethree fetuses injected with the anti-bovine IgM antibody expressed IgM(FIGS. 28C-28E, respectively). As illustrated in FIG. 28F and FIG. 28G,approximately 12.43 to 29.47% of the peripheral blood lymphocytes fromthe control fetuses expressed antibody light chain molecules. For thethree fetuses injected with the anti-bovine IgM antibody, 2.54, 13.77,or 3.99% of the peripheral blood lymphocytes expressed antibody lightchain molecules (FIGS. 28H-28J, respectively). These results indicatethat the injection of the anti-bovine IgM antibody reduced the number ofB-cells in the peripheral blood of the fetuses.

Fetal Cell Transplant Procedures for Tolerization in Ungulates andSubsequent Administration of an Antibody to Inhibit the Production ofEndogenous Antibodies

If desired, ungulate fetuses can be tolerized to proteins or cells fromthe same genus or species used to generate the antibody that is lateradministered to eliminate endogenous antibody. This tolerization shouldreduce or prevent any adverse reaction to the administered, foreignantibody. In one tolerization technique, fetuses in pregnant cows areinjected with a combination of equine marrow cells (2 to 3 mls of 2×10⁷cells/ml) and approximately 1-5 mg of equine serum proteins, such as ofIgM, IgD, IgG, IgE, or IgA on day 75 (2.5 months) of gestation. Thesecells and proteins may be obtained from commercial sources or isolatedusing standard cell purification techniques (such as FACS sorting) orstandard protein purification techniques (see, for example, Ausubel etal., supra). The injection of equine bone marrow cells into the fetusmay be performed by exposing the gravid uterus of the pregnant cow viaflank incision. This procedure is done using appropriate anesthetics andanalgesics. Alternatively, the mouse cells and proteins may beadministered using transvaginal ultrasound, which is minimally invasive.As these cells propagate and integrate into the fetus, tolerance toequine cells is induced in the developing animal.

If desired, one or more fetuses may be recovered during gestation usingstandard Caesarian techniques to determine whether T-cells from thefetus proliferate or produce cytokines in response to equine antigensand to determine whether B-cells secrete anti-equine antibodies.

Alternatively, these equine proteins or cells may be administered afterbirth of the calves. Preferred postnatal routes of administrationinclude parenteral, intravenous, intraarterial, intraventricular,subcutaneous, and intramuscular administration.

The live calves may be immediately injected with an equine antibody(e.g., an anti-IgM antibody) or held until later administration, such asadministration at 1, 2, 4, 6, 8, 10, 12, or 14 months of age. The calvesare injected with equine antibody in one or more sites, and theremaining xenogenous antibodies are purified from blood, milk, or lymphsamples from the calves, as described above. If desired, blood samplesmay also be analyzed to evaluate serum mouse Ig levels, bovine Iglevels, white blood cell levels, and other markers of animal health.

As an alternative to the above method of injecting equine cells into afetus to induce tolerization, equine embryonic cells may be injectedinto a bovine preimplantation embryo to form a germ-line chimera (see,for example, Bradley et al., Nature 309:225-256, 1984). Thepreimplantation embryo may be an embryo in a pregnant cow or an embryothat is cultured in vitro and then transferred to a maternal host, asdescribed below.

EXAMPLE 17 Transgenic Ungulates Having Reducedα-1,3-Galactosyltransferase Activity

If desired, transgenic ungulates in which α-1,3-galactosyltransferase ismutated can be generated to prevent undesired glycosylation ofxenogenous antibodies with a galactose α(1,3)-galactose epitope. Bovinefibroblast cell lines in which one allele of theα-1,3-galactosyltransferase locus is mutated were generated byhomologous recombination. The DNA construct for generating theα-galactosyltransferase knockout cells was used to prevent transcriptionof functional, full-length α-galactosyltransferase mRNA by insertingboth a puromycin-resistance gene (puro, described herein) and atranscription termination cassette (STOP) in exon 9 which contains thecatalytic domain. Thus, the resulting immature α-galactosyltransferasetranscripts lack the catalytic domain. The DNA construct (i.e., theα-galactosyltransferase KO vector) was electroporated into threeindependent bovine fibroblast cell lines, and then puromycin-resistantcolonies were isolated. Based on PCR analysis, homologous recombinationin the exon 9 region occurred in some colonies. Thus, bovine fibroblastcell lines in which one allele of α1,3-galactosyltransferase locus ismutated were generated. If desired, the second allele can be mutated byusing the same knockout vector and a higher concentration of antibioticto select for homozygous knockout cells or using another knockout vectorwith a different antibiotic resistance gene. This method may also beapplied to cells from other ungulates to generate transgenic cells foruse in the nuclear transfer methods described herein to producetransgenic ungulates of the present invention.

These methods are described further below.

Construction of an α-1,3-galactosyltransferase KO vector Theα-1,3-galactosyltransferase KO vector was generated as follows (FIG.23). To isolate genomic DNA around exon 9 of theα-1,3-galactosyltransferase gene, a DNA probe was amplified by PCR usingthe following primer pair 5′-gatgatgtctccaggatgcc-3′ (SEQ ID NO: 61) and5′-gacaagcttaatatccgcagg-3′ (SEQ ID NO: 62). Using this probe, a bovinegenomic λ phage library was screened, and 7 positive λ phage clones wereidentified. One clone, which contained DNA from a male Charolais bovinefibroblast cell, was analyzed further by restriction mapping. The NotI-Xho I genomic fragment containing exon 9 was subcloned intopBluescript II SK(−) in which the Kpn I site had already been replacedwith Srf I, and then both puro and STOP cassettes were inserted at theAvi I site in the Not 1-Xho I genomic fragment which is 5′ to thecatalytic domain. The orientation of both puro and STOP cassettes was asense strand-orientation relative to the α-1,3-galactosyltransferasegene. DT-A diphtheria toxin gene (DT-A, Gibco) was also added to Not Isite of the vector construct. DT-A was inserted in forward orientationrelative to the puromycin-resistance gene in the targeting cassette tokill cells in which the targeting cassette was randomly integrated inthe genome.

Transfection/Knockout Procedures Transfection of three fetal fibroblastscell lines (two from a male Jersey bovine and one from a female Jerseybovine) was performed using a standard electroporation protocol asfollows. The medium used to culture the bovine fetal fibroblastscontained 500 ml Alpha MEM (Gibco, 12561-049), 50 ml fetal calf serum(Hy-Clone #ABL13080), 5 ml penicillin-streptomycin (SIGMA), and 1 ml2-mercaptoethanol (Gibco/BRL #21985-023). On the day prior totransfection, cells were seeded on a T175 tissue culture flask with atargeted confluency of 80-100%, as determined by microscopicexamination. On the day of transfection, about 10⁷ bovine fibroblastscells were trypsinized and washed once with alpha-MEM medium. Afterresuspension of the cells in 800 μl of alpha-MEM, 30 μg of the SrfI-digested DNA was added to the cell suspension and mixed well bypipetting. The cell-DNA suspension was transferred into anelectroporation cuvette and electroporated at 1,000 V and 50 μF. Afterthat, the electroporated cells were plated onto twenty 24-well plateswith the alpha-MEM medium supplemented with the serum. After a 48hour-culture, the medium was replaced with medium containing 1 μg/ml ofpuromycin, and the cells were cultured for 2-3 weeks to select puromycinresistant cells. After selection, all colonies which reached close to100% confluency were picked, and genomic DNA was extracted from thecolonies to screen for the desired homologous recombination events byPCR.

Screening for targeted integrations As described above, the genomic DNAwas extracted from each 24-well independently using the PUREGENE DNAisolation Kit (Gentra SYSTEMS) according to the manufacture's protocol.Each genomic DNA sample was resuspended in 20 μl of 10 mM Tris-Cl (pH8.0) and 1 mM EDTA (EDTA). Screening by PCR was performed using thefollowing primer pair 5′-aagaagagaaaggtagaagaccccaaggac-3′ (SEQ ID NO:63) and 5′-cctgggtatagacaggtgggtattgtgc-3′ (SEQ ID NO: 64). The sequenceof one primer is located in the alpha-1,3-galactosyltransferase KOvector, and the sequence of the other primer is located just outside ofthe integrated vector in the targeted endogenous locus (FIG. 23).Therefore, the expected PCR product should be detected only when the KOvector is integrated into the targeted locus by homologousrecombination.

The PCR reaction mixtures contained 18.9 μl water, 3 μl of 10×LA PCRbuffer II (Mg²⁺ plus), 4.8 μl of dNTP mixture, 10 pmol forward primer,10 pmol of reverse primer, 1 μl of genomic DNA, and 0.3 μl of LA Taq.Forty cycles of PCR were performed by incubating the reaction mixturesat the following conditions: 85° C. for three minutes, 94° C. for oneminute, 98° C. for 10 seconds, and 68° C. for 15 minutes. After PCR, thereaction mixtures were analyzed by electrophoresis. Puromycin-resistantclones which generated PCR products of the expected size were selected(FIG. 23). Thus, bovine fibroblast cell lines in which one allele of theα-1,3-galactosyltransferase locus is mutated by the KO vector weresuccessfully generated.

EXAMPLE 18 Alternative Method for Producing Transgenic Ungulates UsingAdeno-Associated Viruses to Mutate an Endogenous Gene

Adeno-associated virus (AAV) can be used for specific replacement oftargeted sequences present in the genome of cells (Inoue et al., Mol.Ther. 3(4):526-530, 2001); Hirata et al., J. Virol. 74(10):16536-42,2000); Inoue et al., J. Virol. 73(9):7376-80, 1999); and Russell et al.,Nat. Genet. 18(4):325-30, 1998)). The gene targeting rate is highlyefficient in comparison to more conventional gene targeting approaches.AAV has a broad range of host and tissue specificities, includingspecificity for both bovine and human skin fibroblasts. Thus, AAV can beused to produce transgenic ungulate cells containing one or moremutations in an endogenous immunoglobulin (e.g., mu heavy chain, lambdalight chain, kappa light chain, or J chain),alpha-(1,3)-galactosyltransferase, or prion gene. These transgenic cellscan then be used in the nuclear transfer methods described herein toproduce transgenic ungulates of the present invention.

Using AAV resulted in homologous recombination of the bovineimmunoglobulin heavy chain locus at higher frequencies than previouslyobtained using traditional gene targeting strategies (i.e.,electroporation and lipofection procedures). In the first round of genetargeting experiments, five appropriately targeted fibroblast cloneswere obtained out of 73 stable transductants containing the DNAintroduced through an AAV vector.

These experiments were carried out as follows.

AAV Knockout Vectors AAV constructs can disrupt a gene either by simpleinsertion of foreign sequences or replacement of endogenous sequenceswith new sequence present in the AAV vector. FIG. 24 shows an AAVconstruct in which all four coding exons of the bovine immunoglobulinheavy chain mu constant region are present on a 2822 base pairBamHI-XhoI fragment. A 1.16 Kb fragment containing a neomycin resistancemarker present in the commercially available vector, pMC1Neo, wasinserted into a SacII site present in exon 4 of the mu heavy chain locusfrom a Holstein bovine. This locus is the one contained in the phageclone isolated to generate the knockout vector described herein. Togenerate the AVV vector, the SacII site in the mu heavy chain locus wasfilled in to create blunt ends, which were then ligated to blunt SalIlinkers (New England Biolabs). Then, the XhoI fragment of pMC1Neo, whichcontains the neomycin resistance gene, was ligated to the SalI siteadded to the locus through the Sail linker. This ligation can beperformed because the XhoI and SalI restriction sites have compatibleends. This knockout vector causes a disruptional insertion of theneomycin resistance gene into the endogenous mu heavy chain gene,thereby inactivating the mu heavy chain gene. This gene inactivationoccurs without deleting regions of the endogenous mu locus.

An alternative vector was designed to remove exons 3 and 4 from theendogenous locus during targeting, resulting in the replacement of thesetwo exons with a functional copy of the neomycin resistance gene (FIG.25). This construct was generated using PCR amplification of genomic DNAfrom a female Jersey bovine. In particular, the 3′ region of homologywas amplified using the following primers: 5′GGGGTCTAGAgcagacactacactgatgggcccttggtcc 3′ (SEQ ID NO: 65), which addsa XbaI restriction site, and 5′ GGGGAAGCTTcgtgtccctggtcctgtctgacacag 3′(SEQ ID NO: 66), which adds a HindIII restriction site. The 5′ region ofhomology was amplified with primers 5′GGGGCTCGAGgtcggcgaaggatggggggaggtg 3′ (SEQ ID NO: 67), which adds a XhoIrestriction site, and 5′ GGGGGGTACCgctgggctgagctgggcagagtggg 3′ (SEQ IDNO: 68), which adds a KpnI restriction site. The capitalized nucleotidesin these primer sequences are nucleotides that do not anneal to the muheavy chain locus but are included in the primers to add restrictionsites to facilitate later subcloning steps. The first four guanines areadded to separate the restriction sites from the very end of the primersbecause restriction enzymes do not cleave sites that are at the very endof primers as well as internal sites. The 5′ region of homology is 1.5Kb long and contains exons 1 and 2. The 5′ region of homology alsocontains the first 25 nucleotides of exon 3 to maintain the spliceacceptor site of exon 3. The splice acceptor site allows exon 3 to beused for splicing and thus prevents the possible splicing of exons 1 and2 to the downstream transmembrane domain to form an aberrantmembrane-bound product. The 3′ region of homology is 1.24 Kb long andcontains the region immediately downstream of exon 4.

For the construct shown in FIG. 24, the targeting cassette was insertedinto the AAV vector reported by Ryan et al. (J. of Virology70:1542-1553, 1996), which contains viral long terminal repeat (LTR)sequences, using standard methods. The AAV vector was packaged intocapsids using the TtetA2 packaging cell line as previously described(Inoue and Russell, 1998, J. Virol. 72:7024-7031, 1998) and purified aspreviously described (Zolotukhin et al., Gene Therapy, 6: 973-985,1999). For the construct shown in FIG. 25, the above method or any otherstandard method can be used to insert the targeting cassette into theAAV vector described by Ryan et al. or any other AAV vector (such as acommercially available vector from Stratagene) and generate virusescontaining the vector.

Transduction procedures Fibroblasts from a female Jersey bovine wereseeded into one well of a 48 well tissue culture plate at 40,000 cellsper well and cultured in complete medium at 38.5° C. and 5% CO₂ untilcells attached to the bottom surface of the well. Once cells adhered,the medium was removed and replaced with 0.2 ml of fresh mediumcontaining AAV particles with the vector shown in FIG. 24 at amultiplicity of infection (MOI) of 500-20,000 particles/cell. The MOIwas chosen based on pilot experiments that determined the resultingnumbers of colonies and the spacing of the colonies during the drugselection phase. Plates were incubated overnight. After this incubation,the transduced wells were rinsed with calcium and magnesium-free PBS anddetached from the wells using either trypsin or the cell dissociationbuffer described above. A uniform cell suspension was obtained by gentlepipetting of the detached cells, and the cells from the well wereredistributed among ten 100 mm tissue culture dishes. Dishes wereincubated with complete medium overnight.

Following this incubation of the 100 mm dishes, the medium was replacedwith selective medium containing G418 at a concentration of 350micrograms/ml. Selective medium was changed every 2-3 days untilcolonies were macroscopically visible on the surface of the dish. Atthat point, individual colonies were picked and transferred into theirown vessels.

Regions containing colonies were marked on the outer surface of thetissue culture dish. Once all colonies were circled, medium wasaspirated off the plates, and the plates were washed three times withcalcium and magnesium-free PBS. After washing, the plates were floodedwith a 1:25 dilution of 1× trypsin and allowed to sit at roomtemperature until the colonies had visibly begun to detach from thesurface of the plate. Plates were kept stationary to prevent detachedcolonies from floating to another location of the plate. A pipette tipwas used to pick up cell clumps in a volume of 50 microliters, and thecontents of the pipette tip were transferred into one well of a 24 welltissue culture plate. Once all colonies were transferred, completemedium containing G418 was added, and the isolated clones were allowedto proliferate to near confluency.

When an individual well was close to confluency, it was washed twicewith calcium and magnesium free PBS. Cells were detached using 0.2 ml ofcell dissociation buffer. Of this cell suspension, 20 μl was transferredto a new 24 well plate, and the remaining cells were allowed to reattachto the surface of the original 24 well plate following the addition of2.0 ml of complete medium. The original plate was incubated to 100%confluency. The new plate serves as a source of appropriately targetedcells for future bovine cloning procedures.

When a well from the original 24 well plate became 100% confluent, themedium was removed, and the cells were washed once with PBS. PBS wasremoved and replaced with a cell lysis buffer adopted from Laird et al.(Nucleic Acids Res. 19:4293, 1991). Briefly, 0.2 ml of lysis buffercontaining 200 mM NaCl, 100 mM Tris-HCl pH 8.5, 5 mM EDTA, 0.2% SDS, and100 ug/ml proteinase K was added to the well. The plate was returned tothe incubator for between three hours and overnight. The viscous celllysate was then transferred to a microfuge tube. An equal volume ofisopropanol was added to precipitate DNA. Following a 10 minute spin ina microfuge, the supernatant was discarded, and the pellet was washedonce with 0.5 ml of 70% ethanol. After removal of the ethanol, the DNApellet was air-dried and resuspended in 35 microliters of TE buffer (10mM Tris pH 8 and 1 mM EDTA). Aliquots of 3 μl were used for PCRanalysis.

PCR analysis DNA samples from drug resistant clones transduced with AAVparticles were screened for appropriate targeting of the vector usingPCR analysis. This screening strategy used one primer that annealswithin the DNA encoding the drug selection marker and another primerthat anneals within the targeted locus, but outside the sequence presentin the AAV targeting particles. PCR products are only detected if theAAV targeting DNA has integrated into the desired location of theendogenous genome.

Results from a single targeting experiment using these AAV particles areshown in FIG. 26. Based on this analysis, five out of 73 independentclones contained the appropriate targeted vector DNA.

This method may also be used with the AAV vector shown in FIG. 25 orwith any other appropriate adenovirus or adeno-associated viral vector.If desired, the second mu heavy chain allele can be mutated in theisolated colonies by transducing them with an AAV vector with adifferent antibiotic resistance gene (i.e., a gene other than a neomycinresistance gene). To select the resulting homozygous knockout cells, theinfected cells are cultured in the presence of the correspondingantibiotic. Alternatively, the isolated colonies can be transduced withan AAV vector containing a neomycin resistance gene and cultured in thepresence of a high concentration of antibiotic (i.e., a concentration ofantibiotic that kills heterozygous knockout cells but not homozygousknockout cells).

EXAMPLE 19 Testing for Human Ig Expression

Testing calves retaining a HAC or other nucleic acid encoding axenogenous antibody may begin shortly after birth and includesevaluation for (1) xenogenous Ig expression, (2) response toimmunization, (3) affinity maturation, and (4) transmission of the HACsto offspring.

Human Ig expression may be monitored by bleeding the animals andassaying for the presence of human heavy and light chain expression byELISA, RT-PCR, or FACS analysis (see, for example, WO 01/35735, filedNov. 17, 2000). Once it has been determined that the animals producehuman Ig, animals are immunized with tetanus toxoid in adjuvant. Animalsare bled once a week following immunization and responses to antigendetermined via ELISA or FACS and compared to pre-bleeds collected beforeimmunization. One month after the initial immunization, animals areboosted with an aqueous form of the antigen. One week following theboost, the animals are bled and response to antigen is measured viaELISA or FACS and compared to the prebleed. The ELISA or FACS assaypermits measurement of most of the titer of the response as well as theheavy chain isotypes produced. This data allows a determination of anincrease in antibody titer as well as the occurrence of class switching.Estimates of average affinity are also measured to determine if affinitymaturation occurs during the response to antigen.

After the transgenic bovines have been obtained as described above, theyare utilized to produce transgenic Igs, preferably human, butpotentially that of other species, e.g. dog, cat, non-human primate,other ungulates such as sheep, pig, goat, murines such as mouse, rat,guinea pig, rabbit, etc. As noted, Ig genes are known to be conservedacross species.

Transgenic Antisera and Milk Containing Xenogenous Antibodies

The bovine (or other ungulate) yields transgenic antisera directed towhatever antigen(s) it is endogenously exposed, or to exogenouslyadministered antigen(s). For example, antigens may be administered tothe ungulate to produce desired antibodies reactive with the antigens,including antigens such as pathogens (for example, bacteria, viruses,protozoans, yeast, or fungi), tumor antigens, receptors, enzymes,cytokines, etc. Exemplary pathogens for antibody production include,without limitation, hepatitis virus (for example, hepatitis C),immunodeficiency virus (for example, HIV), herpes virus, parvovirus,enterovirus, ebola virus, rabies virus, measles virus, vaccinia virus,Streptococcus (for example, Streptococcus pneumoniae), Haemophilus (forexample, Haemophilus influenza), Neisseria (for example, Neisseriameningitis), Coryunebacterium diptheriae, Haemophilus (for example,Haemophilus pertussis), Clostridium (for example, Clostridiumbotulinium), Staphylococcus, Pseudomonas (for example, Pseudomonasaeruginosa), and respiratory syncytial virus (RSV).

One or more pathogens may be administered to a transgenic ungulate togenerate hyperimmune serum useful for the prevention, stabilization, ortreatment of a specific disease. For example, pathogens associated withrespiratory infection in children may be administered to a transgenicungulate to generate antiserum reactive with these pathogens (e.g.,Streptococcus pneumoniae, Haemophilus influenza, and/or Neissariameningitis). These pathogens may optionally be treated to reduce theirtoxicity (e.g., by exposure to heat or chemicals such as formaldehyde)prior to administration to the ungulate.

For the generation of broad spectrum Ig, a variety of pathogens (e.g.,multiple bacterial and/or viral pathogens) may be administered to atransgenic ungulate. This hyperimmune serum may be used to prevent,stabilize, or treat infection in mammals (e.g., humans) and isparticularly useful for treating mammals with genetic or acquiredimmunodeficiencies.

In addition, antibodies produced by the methods of the invention may beused to suppress the immune system, for example, to treat neuropathies,as well as to eliminate particular human cells and modulate specificmolecules. For example, anti-idiotypic antibodies (i.e., antibodieswhich inhibit other antibodies) and antibodies reactive with T-cells,B-cells, or cytokines may be useful for the treatment of autoimmunedisease or neuropathy (e.g., neuropathy due to inflammation). Theseantibodies may be obtained from transgenic ungulates that have not beenadministered an antigen, or they may be obtained from transgenicungulates that have been administered an antigen such as a B-cell,T-cell, or cytokine (e.g., TNFα).

Transgenic antisera generated from transgenic ungulates that have notbeen administered an antigen may be used to manufacture pharmaceuticalscomprising human polyclonal antibodies, preferably human IgG molecules.These human antibodies may be used in place of antibodies isolated fromhumans as Intravenous Immunoglobulin (IVIG) therapeutics.

Transgenic antiserum may optionally be enriched for antibodies reactiveagainst one or more antigens of interest. For example, the antiserum maybe purified using standard techniques such as those described by Ausubelet al. (Current Protocols in Molecular Biology, volume 2, p.11.13.1-11.13.3, John Wiley & Sons, 1995). Preferred methods ofpurification include precipitation using antigen or antibody coatedbeads, column chromatography such as affinity chromatography, magneticbead affinity purification, and panning with a plate-bound antigen.Additionally, the transgenic antiserum may be contacted with one or moreantigens of interest, and the antibodies that bind an antigen may beseparated from unbound antibodies based on the increased size of theantibody/antigen complex. Protein A and/or protein G may also be used topurify IgG molecules. If the expression of endogenous antibodies is noteliminated, protein A and/or an antibody against human Ig light chainlambda (Pharmingen) may be used to separate desired human antibodiesfrom endogenous ungulate antibodies or ungulate/human chimericantibodies. Protein A has higher affinity for human Ig heavy chain thanfor bovine Ig heavy chain and may be used to separate desired Igmolecules containing two human heavy chains from other antibodiescontaining one or two ungulate heavy chains. An antibody against humanIg light chain lambda may be used to separate desired Ig moleculeshaving two human Ig lambda chains from those having one or two ungulateIg light chains. Additionally or alternatively, one or more antibodiesthat are specific for ungulate Ig heavy or light chains may be used in anegative selection step to remove Ig molecules containing one or twoungulate heavy and/or light chains.

The resultant antisera may itself be used for passive immunizationagainst an antigen. Alternatively, the antisera has diagnostic,prophylactic, or purification use, e.g. for attaining purification ofantigens.

Alternatively, after antisera administration, B-cells may be isolatedfrom the transgenic bovine and used for hybridoma preparation. Forexample, standard techniques may be used to fuse a B-cell from atransgenic ungulate with a myeloma to produce a hybridoma secreting amonoclonal antibody of interest (Mocikat, J. Immunol. Methods225:185-189, 1999; Jonak et al., Hum. Antibodies Hybridomas 3:177-185,1992; Srikumaran et al., Science 220:522, 1983). Preferred hybridomasinclude those generated from the fusion of a B-cell with a myeloma froma mammal of the same genus or species as the transgenic ungulate. Otherpreferred myelomas are from a Balb/C mouse or a human. In this instance,hybridomas are provided that make xenogenous monoclonal antibodiesagainst a particular antigen. For example, this technology may be usedto produce human, cat, dog, etc. (dependent upon the specific artificialchromosome) monoclonal antibodies that are specific to pathogens.Methods for selecting hybridomas that produce antibodies havingdesirable properties, i.e., enhanced binding affinity, avidity, are wellknown.

Alternatively, a B-cell from a transgenic ungulate may be geneticallymodified to express an oncogene, such as ras, myc, abl, bcl2, or neu, orinfected with a transforming DNA or RNA virus, such as Epstein Barrvirus or SV40 virus (Kumar et al., Immunol. Lett. 65:153-159, 1999;Knight et al., Proc. Nat. Acad. Sci. USA 85:3130-3134, 1988; Shammah etal., J. Immunol. Methods 160-19-25, 1993; Gustafsson and Hinkula, Hum.Antibodies Hybridomas 5:98-104, 1994; Kataoka et al., Differentiation62:201-211, 1997; Chatelut et al., Scand. J. Immunol. 48:659-666, 1998).The resulting immortalized B-cells may also be used to produce atheoretically unlimited amount of antibody. Because Ig is also secretedinto the milk of ungulates, ungulate milk may also be used as a sourceof xenogenous antibodies.

EXAMPLE 20 Optional Evaluation of Pain, Discomfort, and Overall Healthof Ungulates

If desired, the ungulates used in the methods of the invention may beevaluated for signs of pain or discomfort from the administeredantibody. Standard clinical chemistry analyses may be performed on bloodsamples from the mammals. White blood cell counts and red blood cellcounts may also be determined and compared to clinical norms.Respiration, heart rate, and temperature are determined on a weeklybasis. Food and water intake is measured. In addition, daily behavioralobservations are made and recorded on a score chart. The score chartincludes observations of activity, watery or dry eyes and nose, andsigns of diarrhea. Periodic estimates (e.g., weekly) of the amount ofxenogenous and endogenous antibody may be made.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication orpatent application was specifically and individually indicated to beincorporated by reference.

1. A transgenic ungulate whose genome comprises a mutation in anendogenous immunoglobulin (Ig) heavy chain or Ig light chain locus, saidmutation reducing the expression of said Ig heavy chain or Ig lightchain.
 2. The ungulate of claim 1, wherein said mutation reduces theexpression of functional IgM heavy chain.
 3. The ungulate of claim 1,wherein said mutation substantially eliminates the expression of said Igheavy chain or said Ig light chain.
 4. The ungulate of claim 1, whereinsaid mutation is hemizygous.
 5. The ungulate of claim 1, wherein saidmutation is homozygous.
 6. The ungulate of claim 1, wherein saidmutation is an insertion of a positive selection marker into saidendogenous locus.
 7. The ungulate of claim 6, wherein said positiveselection marker is an antibiotic resistance gene.
 8. The ungulate ofclaim 7, wherein each allele comprises the same antibiotic resistancegene.
 9. The ungulate of claim 7, wherein each allele comprises adifferent antibiotic resistance gene.
 10. The ungulate of claim 6,wherein said positive selection marker is operably linked to axenogenous promoter.
 11. The ungulate of claim 1, wherein said mutationis an insertion of a transcription termination sequence into saidendogenous loci.
 12. The ungulate of claim 11, wherein saidtranscription termination sequence is inserted downstream of the initialATG codon in exon 2 of an endogenous mu heavy chain locus.
 13. Theungulate of claim 1, comprising one or more nucleic acids comprising oneor more transgenes and expressing an mRNA or protein encoded by saidtransgene(s).
 14. The ungulate of claim 1, the cells of said ungulatefurther comprising one or more chromosomal fragments comprising one ormore, all or part of an unrearranged xenogenous Ig locus which undergoesrearrangement and expresses one or more xenogenous Ig molecules inB-cells.
 15. The ungulate of claim 14, wherein said molecule is anantibody protein.
 16. The ungulate of claim 15, wherein said antibodyprotein is a human antibody protein.
 17. The ungulate of claim 1,wherein said ungulate is a pig.
 18. An isolated ungulate somatic cellcomprising a mutation in an endogenous immunoglobulin (Ig) heavy chainor Ig light chain locus, said mutation reducing the expression of saidIg heavy chain or Ig light chain.
 19. The cell of claim 18, comprising amutation in both alleles of said IgM heavy chain or said light chain.20. The cell of claim 18, wherein said mutation is a transcriptiontermination sequence.
 21. The cell of claim 20, wherein saidtranscription termination sequence is inserted downstream of the initialATG codon in exon 2 of an endogenous mu heavy chain locus.
 22. The cellof claim 18, further comprising one or more chromosomal fragmentscomprising one or more, all or part of a xenogenous Ig locus.
 23. Thecell of claim 18, wherein said cell is a fetal fibroblast or a B-cell.24. The cell of claim 18, wherein said ungulate is a pig.
 25. A methodof producing antibodies, said method comprising the steps of: (a)administering one or more antigens of interest to an ungulate whosegenome comprises a mutation in an endogenous immunoglobulin (Ig) heavychain or Ig light chain locus and the cells of said ungulate furthercomprising a chromosomal fragment or fragments comprising anunrearranged human light chain locus and an unrearranged human heavychain locus, wherein the loci undergo rearrangement resulting in theproduction of human antibody proteins specific for said one or moreantigens; and (b) recovering said human antibodies from said ungulate.26. The method of claim 25, wherein said mutation is an insertion of atranscription termination sequence into said endogenous locus.
 27. Themethod of claim 26, wherein said transcription termination sequence isinserted downstream of the initial ATG codon in exon 2 of an endogenousmu heavy chain locus.
 28. A method of producing antibodies, said methodcomprising recovering human antibodies from an ungulate whose genomecomprises a mutation in an endogenous immunoglobulin (Ig) heavy chain orIg light chain locus and the cells of said ungulate further comprising achromosomal fragment comprising a human antibody gene locus, whereinsaid antibody gene locus undergoes rearrangement resulting in theproduction of human antibodies.
 29. The method of claim 28, wherein saidmutation is a transcription termination sequence into said endogenouslocus.
 30. The method of claim 29, wherein said transcriptiontermination sequence is inserted downstream of the initial ATG codon inexon 2 of an endogenous mu heavy chain locus.
 31. The method of claims25 or 28, wherein said antibodies are directed against a desiredantigen.
 32. The method of claims 25 or 28, wherein said antibodies arepolyclonal.
 33. The method of claims 25 or 28, wherein said antibodiesare recovered from the serum of said ungulate.
 34. The method of claims25 or 28, wherein said ungulate is a pig.
 35. A method for producing atransgenic ungulate having reduced expression of an endogenous Ig heavychain or Ig light chain locus, said method comprising the steps of: (a)incubating a permeabilized cell of claim 18 in an extract from a mitoticsomatic cell or oocyte under conditions that allow chromatincondensation and nuclear envelope breakdown in said permeabilized cell;(b) inserting said cell formed in step (a) into a nucleated orenucleated ungulate oocyte, thereby forming a reconstituted oocyte; and(c) transferring said reconstituted oocyte or an embryo formed from saidreconstituted oocyte into the uterus of a host ungulate under conditionsthat allow said reconstituted oocyte or said embryo to develop into afetus.
 36. The method of claim 35, wherein, prior to step (b), said cellis incubated under conditions that allow the membrane of said cell toreseal.
 37. The method of claim 35, wherein said cell is purified fromsaid extract prior to insertion into said oocyte.
 38. The method ofclaim 35, wherein said fetus develops into a viable offspring.
 39. Themethod of claim 38, further comprising mating two offspring to produce atransgenic ungulate whose genome comprises mutations in both alleles ofan endogenous immunoglobulin (Ig) heavy chain or Ig light chain locus.40. The method of claim 35, wherein said oocyte from step (b) iscultured under conditions that allow cell division and one of theresulting cells is recloned one or more times.
 41. The method of claim35, wherein said permeabilized cell and said oocyte are from the samespecies.
 42. The method of claim 35, wherein said permeabilized cell isa fibroblast, epithelial cell, neural cell, epidermal cell,keratinocyte, hematopoietic cell, melanocyte, chondrocyte, macrophage,monocyte, fibroblast, muscle cell, embryonic stem cell, embryonic germcell, fetal cell, placental cell, a cell of the female reproductivesystem, or embryonic cell.
 43. The method of claim 35, wherein saidmutation in said endogenous locus of said ungulate somatic cell of claim18 is an insertion of a transcription termination sequence into saidendogenous locus.
 44. The method of claim 43, wherein said transcriptiontermination sequence is inserted downstream of the initial ATG codon inexon 2 of an endogenous mu heavy chain locus.
 45. The method of claim35, wherein said ungulate is a pig.